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Theileriosis caused by Theileria annulata can be effectively prevented by vaccination with attenuated, cultured schizonts. Although these attenuated vaccines have been applied for a long time, not much is known about the fate of the vaccine strain in the field. Here, two experimental Spanish vaccine strains originating in Cádiz and Cáceres, and one Sudanese strain are studied to address the development of a carrier status and the infectivity for Hyalomma ticks. Moreover, the heterogeneity of the merozoite surface protein, Tams1, was analyzed in search for an attenuation marker. Using the sensitive reverse line blot (RLB) hybridization, the development of a low level carrier status was demonstrated in the Cáceres and Sudanese line vaccinated calves. Although no signal was detected in the Cádiz line vaccinated calves, seroconversion against the schizont stage was observed, as it was in all other calves. The experimental transmission of T. annulata by Hyalomma ticks to naïve calves was unsuccessful for all cell line inoculated calves. Tams1 heterogeneity indicated a clonal selection of parasites during the process of attenuation, but the Tams1 sequence itself has no connection with the attenuation status. In conclusion, a carrier status develops in attenuated schizont culture vaccinated calves, but is not infective for Hyalomma ticks. Based on these data, the risk for spread of the vaccine strains in the field may be very low.

The major economically important tick-borne diseases of cattle are theileriosis, babesiosis, anaplasmosis, and cowdriosis. Culture-derived attenuated schizonts of Theileria annulata have proved to be safe for all types of cattle and they protect against tick-borne theileriosis. T. parva was also successfully grown in vitro; however, inoculation of cattle with allogeneic schizont-infected cells resulted in rejection and destruction of the parasites together with the host cells. The number of schizont-infected cells needed for immunization is greater than for T. annulata theileriosis. Culture-propagated Babesia bovis and B. bigemina were used for large scale vaccination in the field. An avirulent population of Babesia spp. was obtained by in vitro cloning; inoculation of cattle did not induce clinical babesiosis, but produced specific antibodies. Culture-derived exoantigens of Babesia spp. proved to be completely safe for cattle, however, they conferred less protection than live parasites. Cell-cultured Cowdria ruminantium was highly infective for susceptible animals but, attenuated in vitro, could offer a potential source for vaccination. Anaplasma marginale, successfully grown in tick cell culture, may be developed for vaccines. Factors that should be considered in the developing of vaccines against tick-borne diseases include: the protective immune response to the pathogenic parasite developmental stages, virulence, immunological strain differences, and antigenic variations in cattle and in culture.

Tick borne diseases (TBDs), though neglected, are possibly the major constraints for livestock and agricultural production in southern Italy. The diseases affect mainly the livestock imported from foreign Northern countries. But now, very timidly indeed, the awareness that the use of native breeds could contribute to reduce the ravages of these diseases is growing. Over a period of many centuries Apulian farm animals have naturally developed the ability to thrive in TBDs endemic areas. This trait, which can be defined as tolerance to TBDs is associated with the ability to control parasitaemia and to resist the development of anaemia in the face of infection. It is a matter of fact that the hematological features of Apulian native farm animals are rather peculiar as the authors highlighted by studying the blood of these animals over a period of more than ten years. In the light of their experience the authors considered that the phenomenon of tolerance is a broad-based one and possibly not unrelated to the erythropoietic system of the Apulian animals. Thus the ability to resist anaemia per se was tested in four Altamurana sheep compared to that of four Romanov sheep. This work summarises the results obtained from the experimental anaemization of the above two breed groups and discusses the findings on the basis of a review of the authors' experience in studying TBDs in sheep.

A workshop of the European Union (EU) Concerted Action Project on the Integrated Control of Ticks and Tick-borne diseases (CA-ICTTD) recently assessed protective immune mechanisms to ticks and tick-borne diseases. The consensus achieved there is summarized here by Patricia Preston and Frans Jongejan. The current understanding of this field is expanded upon by the accompanying articles, and Poster, in this special issue of Parasitology Today.

The first experimental vaccination against ticks was carried out 60 years ago. Since then, progress has been slow, although the recent commercial release of a recombinant vaccine against Boophilus microplus is significant. The nature of naturally acquired protective immunity against ticks is poorly understood, particularly in the important, domesticated ruminant hosts. Characterization of the antigens of naturally acquired immunity remains limited, although more has been achieved with 'concealed' antigens. Crucial questions remain about the true impact of tick-induced immunosuppression and the effect of immunity on the transmission of tick-borne diseases, despite some fascinating and important recent results, as discussed here by Peter Willadsen and Frans Jongejan.

Immunological interactions at the tick host interface involve innate and specific acquired host immune defenses and immunomodulatory countermeasures by the tick. Tick feeding stimulates host immune response pathways involving antigen-presenting cells, cytokines, B-cells, T-cells, circulating and homocytotropic antibodies, granulocytes, and an array of biologically active molecules. In response to host immune defenses, tick-mediated host immunosuppressive countermeasures inhibit: host antibody responses; complement activation; T-cell proliferation; and cytokine elaboration by macrophages and Th1-lymphocytes. Immunosuppressive proteins identified in tick salivary glands and saliva have been partially characterised. Tick-induced host immunosuppression facilitates blood meal acquisition and is an important factor in the transmission/establishment of the tick-borne disease-causing agent, Borrelia burgdorferi. A novel strategy for control of tick-borne pathogens is proposed.

Purpose: To determine the prevalence of serologic reactivity, the 1-year incidence of seroconversion, and the frequency of multiple infections, and their associations with symptoms in a group of volunteers at high risk for tick-borne infections in New York state., Methods: We performed a seroepidemiologic study of Lyme borreliosis, 2 of the ehrlichioses, Rocky Mountain spotted fever, and babesiosis among 671 participants who lived or worked in a high-risk area (mainly in eastern Long Island, New York) for tick-borne diseases. Sera were collected in the winters of 1994 and 1995. Signs and symptoms of tick-borne disease were monitored monthly by mail and telephone. Lyme borreliosis serologies were done by enzyme-linked immunosorbent assay and Western blot. Rocky Mountain spotted fever serologies were initially screened using Dip-S-Ticks, followed by specific indirect immunofluorescence. Ehrlichiosis serologies were determined by epifluorescent microscopy, as were antibodies to Babesia microti., Results: Of the 671 participants, 88 (13%) had antibodies to > or = 1 tick-borne organisms, including 34 (5% of the total) with antibodies to Borrelia burgdorferi. Twenty-seven participants had evidence of exposure to B. burgdorferi at baseline. Seven participants (1%) seroconverted during the course of the study, 5 of whom were symptomatic for Lyme borreliosis. Antibodies to spotted fever group rickettsiae were seen in 28 participants (4%), 22 of whom were positive at baseline and 6 of whom seroconverted during the observation period. None of the seropositive patients had any symptoms or signs of infection. Twenty-four participants (3%) had serologic evidence of exposure to Ehrlichia (all but one to Ehrlichia equi); 5 (0.7%) seroconverted during the observation period, including 3 subjects who were asymptomatic. Antibodies to B. microti were seen in 7 participants (1%), including one asymptomatic seroconversion during the year of observation. There was evidence of possible dual infection in 5 patients., Conclusion: In a high-risk population, there was evidence of exposure to 5 tick-borne pathogens; however, many infections were asymptomatic, and coinfections were rare.

We studied sera from patients who had participated in a prospective study of borreliosis in Sweden and had acquired tick bites in areas of the country with a high prevalence of granulocytic ehrlichial infections in animals. The sera were examined for IgG anti Ehrlichia antibodies by an indirect immunofluorescence assay using a locally isolated bovine Ehrlichia antigen. Confirmation of the serological results was done at the Unité des Rickettsies, Marseille, France. Three out of 37 of the investigated patients and 1 out of 100 investigated healthy blood donors had significant antibody titres to granulocytotropic Ehrlichiae. No patient or blood donor had specific antibody titres to Ehrlichia chaffeensis. These data suggest that Scandinavian Ehrlichia species can infect and evoke immunological response in tick-exposed humans.

Experimental transmission of Hepatozoon americanum to dogs was attempted with four ixodid ticks, viz., Rhipicephalus sanguineus, Amblyomma americanum, Amblyomma maculatum and Dermacentor variabilis. Ticks that dogs ingested included some that were laboratory-reared and experimentally fed as nymphs on a dog with naturally occurring hepatozoonosis; other ticks were collected as replete or partially engorged larvae, nymphs and adults from dogs that had hepatozoonosis and natural infestations of ticks. Whole ticks used to expose susceptible dogs orally were partially dissected to help release oocysts. Among eight dogs exposed, only the three that were fed A. maculatum adults experimentally acquisition fed as nymphs became infected. Dogs developed elevated body temperature and other evidence of clinical disease starting 4 weeks after exposure. 'Cysts' typical of H. americanum were found in skeletal muscle when samples were first examined 5 weeks after dogs ingested ticks, and parasites were also observed in peripheral blood smears at approximately the same time. Our study demonstrates that A. maculatum nymphs can acquire H. americanum by feeding on a parasitemic dog and that transstadial transmission of the protozoan occurs, with dogs acquiring infection when they ingest newly molted adult ticks.

In the first type of mutual relationships the ixodid ticks expose an evident pathogenicity to vertebrate hosts (tick toxicoses) and in the second type the hosts expose a resistance on a base of organism immune system activisation in a response to blood sucking. In the third type of relationships an interaction between the ticks and hosts have a dynamic character (tolerability of host). The first type of relationships leading to a selective elimination of hosts according to our opinion means a comparatively youth of relationships. The third type represent ancient and long term relationships. The second type probably having been widely spread in a historical past occupies an intermediate position. Depending upon areal the ticks of the same type expose different degree of the pathogenicity. Even an acquired resistance of the host does exist in natural conditions, it is a transient phenomenon.

Serological tests for Cowdria ruminantium infection have been hampered by low specificity. Here, an indirect ELISA based on purified antigen, a competitive ELISA using a recombinant major antigenic protein (MAP-1) and an indirect ELISA based on the MAP-1B region of the recombinant MAP-1 were compared. The tests were validated using 3000 sera of ruminants from 14 islands of the Lesser Antilles as well as sequential serum samples from 10 cattle, 17 goats and 10 sheep vaccinated with inactivated C. ruminantium in ISA 50 adjuvant and from 14 goats infected with a virulent culture supernatant. All tests detected significantly higher percentages of positives on Antigua, Guadeloupe and Marie-Galante, where C. ruminantium had been isolated before. Overall specificity calculated with sera from the other 11 heartwater-free islands was 98.1%, 98.5%, and 99.4% for the ELISA based on crude antigen, recombinant MAP-1 and MAP-1B, respectively. Sensitivities observed with sequential serum samples were similar for all tests. Tests based on recombinant antigens, especially the MAP-1B, showed improved specificity, suggesting their use for epidemiological studies in regions where the distribution of cowdriosis is unknown. In addition, the competitive ELISA is useful for studies in wildlife for which species-specific conjugates do not exist.

Glycerol preserved, frozen tick-borne disease vaccine strains developed in Australia were imported into Paraguay to test their safety in pregnant Holando heifers and their efficacy against challenge from inoculated local field strains of Babesia bigemina, B. bovis and Anaplasma marginale in Hereford X Criolla heifers. The two Babesia strains proved to be safe and the B. bovis K strain was very effective in providing immunity to a local field strain of B. bovis. The B. bigemina efficacy trial was inconclusive, possibly due to the avirulent nature of the local field strain used in challenge. The A. centrale strain did not prove to be as safe as would be desirable in safety trials, neither did it provide as good protection as the Babesia strains in the efficacy trial. It was concluded that the Babesia strains provided good protection against field challenge in Paraguay and were safe to use in highly susceptible cattle, however an alternative to A. centrale should be sought to provide protection against local strains of A. marginale.

Canine hepatozoonosis is a disease caused by the tick-borne protozoan Hepatozoon canis. Five puppies were inoculated by ingestion of Rhipicephalus sanguineus ticks experimentally infected with H. canis, and all became infected with H. canis: gametocytes were detected in blood smears from four dogs and schizonts were observed in the spleen and bone marrow of the fifth. Antibodies reactive with H. canis gametocytes were detected by the indirect fluorescent antibody test (IFA), with IgM detected initially in all dogs 16 to 39 days post infection (PI) and IgG 22 to 43 days PI. The presence of gametocytes was first observed within peripheral blood neutrophils in Giemsa-stained blood smears between days 28 and 43 PI. Gametocyte-reactive antibodies were detected before the appearance of blood gametocytes in three of the four parasitemic dogs and also in a dog with no observed parasitemia. The detection of serum antibodies prior to the detection of blood gametocytes, or without apparent parasitemia, suggests that antibodies reactive with gametocytes may be formed against earlier forms of the parasite developing in the parenchymal tissues. Sera of dogs experimentally infected with Babesia canis, Babesia gibsoni and Ehrlichia canis exhibited no reactivity when tested with H. canis antigen. Additionally, sera positive for H. canis were not reactive with antigens of Toxoplasma gondii, Neospora caninum, Leishmania donovani and E. canis. In conclusion, incoculation of dogs with ticks infected with H. canis results in production of antibodies reactive with peripheral blood gametocytes. Detection of IgG titres would be beneficial for the diagnosis of progressive infections with undetectable parasitemia, for seroprevalence studies, and as an adjunct to IgM titres in early infections.

Five lambs were inoculated with a granulocytic Ehrlichia species originally isolated from a Swedish horse with granulocytic ehrlichiosis (EGE). The 16S rRNA gene sequence of the Swedish Ehrlichia sp. causing EGE was identical to the sequence of the agent causing human granulocytic ehrlichiosis (HGE). After the inoculation, infected neutrophils and a low serologic response were seen in all lambs, but no clinical symptoms were observed. In one lamb 17% of the neutrophils were infected without a corresponding fever. Six weeks later the lambs were inoculated with an ovine isolate of E. phagocytophila. After challenge with E. phagocytophila the lambs reacted with fever and infected granulocytes. The results presented herein show that the equine Ehrlichia isolate was infective for lambs but generated weak immune response and no distinctive protection from subsequent challenge with E. phagocytophila.

Three adult patients (one woman and two men) presented with polymorphous skin manifestations caused by pigeon ticks (Argas reflexus). These manifestations were characterized by wheals with a central hemorrhagic point in the first patient, crusted-ulcerative lesions in the second, and pustular lesions in the third patient. We observed specific IgE against a somatic extract (antigen A) and the saliva (antigen B) of A. reflexus in two patients.

Serum samples from Peromyscus leucopus (white-footed mouse), collected in Connecticut (USA) in 1983, 1985, and during 1990 to 1993, were analyzed by an enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent antibody (IFA) staining methods for antibodies to Borrelia burgdorferi (strain 2591), Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain). Of the 294 serum samples tested, 160 (54%) contained immunoglobulins to one or more of these pathogens. There were antibodies to two or more etiologic agents in 77 (48%) of the seropositive mice. Although it was uncommon to detect coexisting antibodies to all four pathogens (n = 5 positive mice), E. chaffeensis-reactive antibodies or immunoglobulins to E. equi were present along with those produced to B. burgdorferi and B. microti in 24 other mice. These rodents carry antibodies to several tick-borne pathogens at numerous sites in Connecticut and may play a role in the epizootiology of ehrlichiosis as well as babesiosis and Lyme borreliosis.

In recent years, investigators have gained an increasing appreciation of the complexity of the Lyme disease transmission cycle with regard to the number of pathogens involved. Babesia microti, a blood parasite that is related to the organism that causes malaria, frequently accompanies the Lyme disease spirochete in the mouse reservoir. Recently, a newly described Ehrlichia species related to Ehrlichia equi has been found to be transmitted by the deer tick. Human infections with these agents alone and in combination are now being described, and the successful treatment of these infections may depend on proper diagnosis. The convergence of these and other organisms on the Lyme disease transmission cycle provides a unique opportunity to study pathogen-pathogen interactions in a naturally occurring model.

A seroprevalence and risk factor study of emerging tickborne infectious diseases (Lyme disease, ehrlichiosis, and babesiosis) was conducted among 230 residents of a semirural community in Sonoma County, California. Over 50% of residents reported finding a tick on themselves in the preceding 12 months. Samples from 51(23%) residents were seroreactive to antigens from one or more tickborne disease agents: 1.4% to Borrelia burgdorferi, 0.4% to Ehrlichia equi, 4.6% to Ehrlichia chaffeensis, and 17.8% to the Babesia-like piroplasm WA1. Only 14 (27%) of these seroreactive residents reported one or more symptoms compatible with these diseases. Seroreactivity was significantly associated with younger age (<16 years), longer residence in the community (11-20 years), and having had a physician's diagnosis of Lyme disease. In northern California, the risk of infection with these emerging tickborne diseases, particularly in children, may be greater than previously recognized.

The prevalence of Babesia bovis antibodies was estimated by using an ELISA (98% sensitivity and 95% specificity). Sera were obtained from 165 calves (mean age and standard deviation: 9.7 +/- 2.7 months) from an area in Argentina known to be unfavourable for the development of the vector tick, Boophilus microplus. The area comprised about 300,000 ha used for cattle breeding. The cattle population of 55,000 included 12,000 cattle under 1 year of age. Cattle were maintained mainly on natural grasses in communal lands. The true prevalence of antibodies to Babesia bovis was 12.2% with a confidence interval of 7.6% to 18.2%, and an inoculation rate (h; daily probability of infection) of 0.0004. This confidence interval has its lower boundary in the area of endemic stability due to low h of Babesia bovis by the vector tick and the upper limit in the area of endemic instability. This type of analysis could help to decide the implementation of preventive measures (e.g. vaccination) rationally, even in remote areas (such as the one of the present study) with an extensive cattle industry.

The most important tick-borne disease of cattle in eastern, central and southern Africa is East Coast fever (ECF) caused by Theileria parva and transmitted by the tick Rhipicephalus appendiculatus. Other less-important tick-borne diseases in cattle are benign theileriosis caused by Theileria mutans, babesiosis caused by Babesia bigemina, anaplasmosis caused by Anaplasma marginale and cowdriosis caused by Cowdria ruminatum. In Murang's District, Central Province of Kenya, five agroecological zones (AEZs) are defined according to climate, altitude and agricultural activities. A cross-sectional serological study was conducted on 750 smallholder dairy farms in Murang's District, selected in a stratified random sampling method. The farms had a total of 362 calves. One hundred and fifty farms were studied from three administrative sublocations in each of the five AEZs. Prevalence of serum antibodies to three tick-borne parasites, that is T. parva, T. mutans and B. bigemina, were determined using the enzyme-linked immunosorbent assay (ELISA) technique. Antibody prevalence values differed across the AEZs. The ranges of means for the prevalences were: T. parva (18-72%), T. mutans (1.5-28%) and B. bigemina (12-49%). The above results serve as indicators of the possible existence of endemic stability in some AEZs for some parasites.

A survey for the prevalence of antibodies to Hepatozoon canis and for intraneutrophilic H. canis gametocytes in the peripheral blood neutrophils of dogs in Israel showed that 33.1% were seropositive, while only 1% of the dogs sampled had detectable parasites in their blood smears. Exposure to H. canis is widespread but it appears that most infected dogs undergo a subclinical infection and only a small proportion develop clinical disease.

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.

One hundred and fifty-one forestry workers and 151 matched office clerks were compared as to the presence of antibodies against Borelia burgdorferi, tick-borne encephalitis virus, Puumalavirus and lymphocytic choriomeningitis virus. Their occupational risks of being infected by Borrelia was fourfold and significant, by Puumalavirus and lymphocytic choriomeningitis virus was increased but not significant. No seropositivity has been established against tick-borne encephalitis virus.

The causative agent of Lyme disease, Borrelia burgdorferi, is transmitted by ticks of the Ixodes ricinus complex. In this study, we report the antibody response of recombinant inbred strains of mice of the H-2, b, d, and k haplotypes, infected with B. burgdorferi as a result of exposure to infected I. dammini. The patterns of antibody response assayed by Western blot analysis indicate significant major histocompatibility complex (MHC) restriction to bacterial antigens within the first 2 mo of infection in mice. Other bacterial antigens induce a significant response across the MHC haplotypes tested when assayed on the same bacterial strain used to transmit the infection, but do not crossreact with the same proteins derived from heterologous strains of B. burgdorferi. No response to outer surface protein A was detected at any time during the 60-d period we analyzed this infection. A third group of bacterial antigens appear to generate a MHC-nonrestricted response, and this lack of restriction is maintained when assaying the crossreactivity of the response with other strains of B. burgdorferi. These proteins may provide more accurate diagnostic probes than those currently in use. Finally, there appears to be a significant difference in the expression of most bacterial antigens when the spirochete is cultured for many passages since the same strain of bacterium isolated from low-passage and high-passage preparations exhibit different banding patterns in Western blots when assayed with the same sera.