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We report the increased prevalence recorded in recent years of Rickettsia aeschlimannii in Hyalomma marginatum marginatum ticks removed from human subjects in Castilla y León (NW, Spain). Additionally, a simultaneous infection with R. aeschlimannii and Anaplasma phagocytophilum in two H. marginatum marginatum ticks is reported for the first time.

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Although there are very few reports of human anaphylaxis induced by tick bites, two such cases have recently been seen in Salamanca, Spain. To identify the tick species responsible, salivary-gland extracts from six species of hard tick and two of soft tick were prepared and used as allergens/antigens in skin-prick tests and serological analyses. For each case, the results of the skin tests were positive for several species of hard tick but negative for the soft ticks. ELISA and western blots revealed high titres of IgG against hard ticks (but not soft ticks) in the sera from both cases. However, serum from only one of the cases was found to be ELISA- and western-blot-positive for tick-specific IgE. Accordingly, the anaphylaxis seen in one case was IgE-mediated whereas that in the other case appeared to be IgE-independent. In both cases, most of the tick-specific antibodies only recognized carbohydrate epitopes. High levels of cross-reactivity between the salivary-gland extracts from several species of hard tick made it impossible to identify which species was responsible for each anaphylactic reaction, although the immunological results seem to point to Ixodes ricinus.

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Twenty unfed larvae of Neotrombicula autumnalis (Acari: Trombiculidae) collected on vegetation in the north of Spain were examined by polymerase chain reaction for Borrelia burgdorferi (s.l.). rickettsiae, and the Ehrlichia phagocytophila genogroup. At least 10% of the larvae were found to contain granulocytic ehrlichiae. Because the larvae were unfed, they would necessarily have inherited the bacteria through a transovarian transmission pathway.

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To enhance the specificity and sensitivity of serological detection of swine exposed to Ornithodoros erraticus or O. moubata, we purified the 158, 186, 215 and 260 kDa antigens from the former species and the designated (owing to their MW and charge) 19C, 17A, 20A1 and 20A2 antigens of the latter by HPLC and gel electroelution methods. All the O. erraticus antigens share epitopes and are difficult to purify individually by reverse phase and ion-exchange chromatography due to their molecular similarity. Tested individually by ELISA, all of them give the same optical densities (OD) with anti-O. erraticus sera, and these ODs are always lower with anti-immature than with anti-adult sera. Although immature and adult specimens have the same antigens, immature forms induce more anti-carbohydrate antibodies than adults. This is the reason for the lower ODs of the anti-immature sera against purified antigens, since these latter antigens essentially react with anti-peptide antibodies (hence, increasing the specificity and sensitivity of the serology). The N-terminus of the 260 kDa antigen shows 80-90% similarity with the hemoglobin alpha-chain of many mammals. The antigens of O. moubata are proteins that are very different from one another and are, therefore, readily purified by ion exchange chromatography. The 20A1 antigen appears to be the most immunogenic and is recognized equally by anti-immature and anti-adult sera. This antigen does not give false positive reactions with the negative control sera analyzed and its N-terminus region shares 46.2% homology with the alpha-chain of the C3 component of rabbit complement.

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We analysed in mice why the salivary gland extract (SGE-2) from Ornithodoros erraticus and O. moubata induce a protective response with Freund's adjuvants (FAs) in swine while the saliva, in natural conditions, does not. Such protection has been ascribed to the fact that administration of SGE-2 plus FAs permits the recognition of certain salivary components that under natural conditions are not immunogenic. The present findings confirm this hypothesis since in mice, which are unable to recognize the above components, the SGE-2-FAs do not induce any protection. We rule out the possibility that the cause of this could lie in the absence of prostaglandin E2 in the SGE-2 (vs saliva) since it is not present in either fluid. Neither could it be due to a change in antibody isotype since those induced by parasites bites and by the SGE-2-FAs are the same (IgG2a > IgG1 > IgG2b; not IgG3, IgM, IgE). No IgG2a were seen when the SGE-2 were administered alone or with alum or ricin. It is therefore suggested that first responses would be Th1 and the second ones Th2, although no IgE is seen in the latter responses either. The parasites do not require complement to feed; by contrast, they block its activation and skin cellular infiltrates, such as those elicited by IL-8, MCP-1 and C5a, do not affect them, regardless of the presence or not of antitick antibodies.

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We show by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot that the composition of the soluble extracts of salivary glands (SGE-2) of Ornithodoros erraticus and Ornithodoros moubata is similar to that of the saliva (pilocarpine-induced), and that the extracts are a valid source of antigens for the detection of anti-argasid antibodies. It is also shown that the SGE-2s do not vary qualitatively with the developmental stage, physiological status, or sex of the ticks. The antigenic components (at least in O. erraticus) are released into the SGE-2 by the action of enzymes that can be inhibited by phenylmethane sulfonyl fluoride plus EDTA. Most of the components of the SGE-2, except the antigenic ones, are strongly glycosylated. Accordingly, the deglycosylation of the SGE-2s does not affect the recognition of antigenic components by anti-tick sera. In both species, the major components of the SGE-2s or the saliva are not recognized by the corresponding antisera. These nonimmunogenic components could have vaccinal value but not diagnostic interest. Finally, it is shown that the antigens of O. erraticus and O. moubata do not cross-react with one another and that those of the first species are more antigenic than those of the second.

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On pig farms, elimination of the argasid ticks acting as reservoirs and vectors for African swine fever greatly favours the eradication of this disease. The elimination of Ornithodoros erraticus involves many problems, most of which could be easily solved by the development of an anti-O. erraticus vaccine. With a view to developing this vaccine, we have tested the protective value of the immune response induced in swine by seven 'concealed' antigens and one soluble salivary gland extract. The latter extract was also prepared from Ornithodoros moubata specimens and tested against this tick. Our results indicate that the immune response elicited by the concealed antigens has no protective value against O. erraticus. The immune response induced by the salivary gland extracts against adults of O. erraticus and O. moubata was apparent in a reduced ingestion of blood (40-60%; P < 0.01) (except in males of O. erraticus) and in a significant decrease (40-60%; P < 0.01) in fecundity in 100% of the females of both species. The good results obtained with salivary antigens, which in situations of natural contact have no protective value, are attributed to the fact that when these antigens are injected with adjuvants, the immune system recognizes certain salivary components (probably those which enable the parasite to feed) which it does not recognize under natural conditions of exposure.

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For some time it has been known that the tick Ornithodoros erraticus might be one of the causes of the persistence of African swine fever in the Iberian Peninsula since its introduction in 1960. In the province of Salamanca serological methods have been used to study the relationship between the presence of the tick in different townships and the outbreaks of African swine fever in these townships between 1987 and 1992. The results showed that there was a statistically significant association between the presence of the parasite and the persistence of African swine fever. In townships without O erraticus traditional methods of control are in most cases enough to avoid new outbreaks of the disease, but in areas with O erraticus the traditional methods should be reinforced by other methods for preventing contact between pigs and the parasite.

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Ornithodoros erraticus is the European vector of African Swine Fever. It is therefore essential to know on which pig farms the tick is present in order to prevent contact with swine. Currently, studies are being made to ascertain this through the detection of anti-O. erraticus antibodies in the sera of swine, using three extracts from the salivary glands of the parasite (SGE): a complete extract (SGE-1), a soluble antigens extract (SGE-2), and a tissue antigens extract (SGE-3). The results of the present work show that SGE-2 gives the best differentiation between swine bitten by O. erraticus and unbitten swine in the enzyme-linked immunosorbent assay (ELISA). Using this extract, an optical density (OD) five-fold higher than the basal OD indicates that the pigs carry anti-O. erraticus antibodies. A serological study carried out in Salamanca with 8083 sera from 1756 pig farms revealed the presence of the parasite on 135 farms. However, during this study we noticed that some sera of unbitten animals gave false-positive reactions. Western blot analysis of SGE-2 of these false-positive sera demonstrated the same bands (except for two) as the real anti-O. erraticus sera. We observed, in ELISA and Western blot analysis, that such false-positive sera only recognised carbohydrate epitopes on SGE-2. This reactivity disappeared on deglycosylated SGE-2 (SGE-2-P). Therefore, SGE-2-P is the antigen that confers the greatest specificity to serology. In this study it was also observed that the low levels of anti-O. erraticus antibodies found in some cases may be because the swine were bitten some months previously on a different farm or that the current farm harboured only a few specimens of O. erraticus, so pig-tick contact is unlikely and hence the pigs either only develop a primary response or the time between contacts is very long and the levels of antibodies fall. Since pigs could be bitten on a different farm, the presence of low levels of anti-O. erraticus antibodies in pig sera do not necessarily indicate the presence of the tick on the farm where sampling was carried out.

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In Spain, considerable efforts are currently being devoted to the eradication of Ornithodoros erraticus from the swine farms harbouring this parasite, the European vector of African swine fever (ASF). However, to do so, a preliminary requirement is to determine on which farms it is present. Of all possible methods for discovering this, the only one feasible for large scale application is the serological detection of swine bearing anti-O. erraticus antibodies. To apply serology it was necessary to check the specificity of extracts from the salivary glands (SGE) from O. erraticus. For this, indirect ELISA, competitive ELISA and Western blot were used to assay the SGE from O. erraticus and their corresponding antisera against the SGE and respective antisera from 4 ixodidae, one mange mite, one louse and a mosquito. The results obtained show that only the anti-ixodidae sera are able to react against the SGE from O. erraticus. The cause of this reaction are the somatic antigens present in the SGE of the argasid but not its soluble (secretory) antigens. It is proposed that the anti-cement antibodies present in the anti-ixodidae sera are those that react with the somatic antigens of O. erraticus.

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To discover whether the immune system of Iberian-breed pigs exerts any adverse action on Ornithodoros erraticus, 3 pairs of pigs were subjected to a weekly infestation over 12 wk with 1,000 larvae, 500 nymphs-1, or 200 adults. Each pair was bitten by only 1 developmental stage. Batches of parasites identical to the foregoing ones were fed weekly on control swine. In none of the 10 parameters studied for each of the batches fed weekly was any significant difference found that could be attributed to the state of sensitization of the animals in which, in a previous study, the presence of high titers of anti-O. erraticus antibodies was found. It was observed that the possible pruritus due to immediate hypersensitivity reactions, which in the test animals appeared after the third week, had no protective value in the natural milieu. In view of the inability of the swine to exert any control over the soft ticks, it is concluded that the size of their populations in the pig pens and their composition according to the developmental stage are factors that depend exclusively on the opportunities that swine breeders offer such populations to feed on the animals.

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Ornithodoros erraticus was found in 30.7 per cent, 35.0 per cent and 71.0 per cent of the pig-pens sampled in the provinces of Salamanca, Badajoz and Huelva in which African swine fever is a problem in the rearing of Iberian pigs. Between 38 and 65 per cent of the pig-pens in these areas are now abandoned and their populations of O erraticus are extinct or becoming so because they can no longer feed on pigs, which in Spain are their main hosts. The abandonment of pig-pens has resulted in the elimination of most soft ticks infected with the virus of African swine fever, and means that the distribution of ticks is now irregular and focal. Another factor affecting their distribution is the kind of soil on which the pig-pens are located. In abandoned pig-pens, the adults and large nymphs survive for about five years or longer when animals occasionally enter them. Hungry tick populations may transmit African swine fever when feeding in winter, whereas the populations that have continuous access to pigs do not feed until the pig-pens reach a temperature of 13 to 15 degrees C. In the latter populations, each stage exhibits a single annual peak of activity, which implies that the development from larva to adult takes two to three years. Pigs may die as a result of the bites, but on no occasion were 100 per cent of the fasting ticks seen to feed, even though they had the opportunity of doing so. This may hinder the eradication of this soft tick from infested pig-pens.

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Larval susceptibility to temephos, malathion and fenitrothion were studied in 6 Aedes, 2 Culex and one Anopheles species from areas with the following agricultural characteristics: VdT, intensively irrigated crops; Zone A, cereals and some irrigation; Zone B, cereals and grasslands; Zone C, oak and pine forests. The susceptibility of adult Anopheles from VdT and Zone B areas to dieldrin and DDT was also studied. The use of insecticides in these areas probably decreases from VdT, to Zone A, to Zone C, to Zone B. Tests of larval and adult susceptibility of mosquitoes in these areas followed the recommendations of WHO re materials and methods. In the absence of any baseline data concerning the susceptibility of the tested mosquito population, the test results only allow one to suspect changes in susceptibility to temephos, malathion and fenitrothion in larvae of An. atroparvus and Cx. theileri from VdT and resistance in adults of An. atroparvus to dieldrin and DDT.

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Of 433 dogs examined in 4 natural zones of Salamanca Province, Spain, the prevalence of canine leishmaniasis (IFAT titres greater than or equal to 1/80) was 0-3% in most mountain villages and from the Castillian plateau (greater than 600 m.a.s.l.) and 10-15% in villages situated on hillsides ("flanc du coteau") with elevations less than 600 m.a.s.l. An exception was the high prevalence found in the area around the city of Salamanca owing to its height (800 m.a.s.l.) and flood-plain situation. Of 5,105 sandflies captured with sticky traps, P. perniciosus and P. ariasi were well represented at the sites with high prevalences of canine leishmaniasis but not elsewhere. Their abundance in the periurban area of the city of Salamanca is attributed mainly to human transformation of the environment. Of the two species, only with P. ariasi can a linear relationship be observed between density and the prevalence of canine leishmaniasis, which seems to show that in Salamanca P. ariasi is its main vector. The probable lower observed vectorial capacity of P. perniciosus could be due to the fact that no progressive physiological ageing towards the end of the summer was observed. Patients with leishmaniasis in the Province come from sites with the highest prevalence of canine leishmaniasis.

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By sporadic examination of 492 wild animals and regular weekly checks of dogs (179), cattle (288) and sheep and goats (141), 13 Ixodides were identified in the province of Salamanca. Some of these were only detected on the northern (I. ricinus) or southern (H. lusitanicum and B. annulatus) slopes of the mountains to the south of the province. Except for occasional introductions, H. lusitanicum and B. annulatus are not found on the Castillian plateau. In wild animals, 12 species were found of which only 4 are monotropic for some of these animals. In dogs, the most important species were R. turanicus and R. sanguineus. The latter shows only one generation per year and its nymphs are held to be the most important vector for the cases of boutonneuse fever registered in the provincial setting. In cattle, R. bursa is the most important tick, in terms of its prevalence in June/July (adults) and from November to January (immature forms). It also displays a much higher parasitization intensity than that of any of the other 7 species found on cattle. These remaining tick species (D. marginatus, H. marginatum, H. lusitanicum, I. ricinus, R. turanicus, Hae. punctata and B. annulatus) are abundant when R. bursa is absent; that is, in spring and at the end of summer, which thus are periods of richness in Ixodides but of low parasitism intensity. The ticks found on sheep and goats are the same as those detected on cattle, but in some of them prevalence and intensity vary as result of the kind of pasture frequented by the ovine and caprine hosts.

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