Kinetics of Experimental Infection of Sheep with Ehrlichia ruminantium Cultivated in Tick and Mammalian Cell Lines

Inoculation of sheep with Ehrlichia (previously Cowdria) ruminantium which has been cultivated in mammalian endothelial cell cultures is almost always followed by a severe clinical reaction, whereas inoculation of the agent cultivated in tick cell lines usually does not provoke a clinical response, but may result in seroconversion and/or protection against subsequent challenge with virulent stabilates. A quantitative, real-time PCR assay was developed to determine the kinetics of infection (rickettsaemia) in sheep inoculated with tick cell- and mammalian cell-derived E. ruminantium (Gardel isolate). The method and initial results are described, and the significance of the findings is discussed in relation to the clinical responses of the sheep. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular detection of <Emphasis Type="Italic">Ehrlichia ruminantium</Emphasis> infection in <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> ticks in The Gambia

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3–15.1%) than in the easterly divisions (Central River and Upper River; range 1.6–7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.     

Prevalence of Ehrlichia ruminantium in adult Amblyomma variegatum collected from cattle in Cameroon

Ehrlichia ruminantium, the etiologic agent of the economically important disease heartwater, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, particularly A. hebraeum and A. variegatum. Although serologic and microscopic evidence of the presence of heartwater have been reported in ruminants in Cameroon, knowledge of E. ruminantium infection in the tick vector, A. variegatum, is lacking. In order to determine the infectivity of A. variegatum ticks by E. ruminantium, we analysed 500 un-engorged A. variegatum ticks collected by hand-picking from predilection sites from 182 cattle [115 ticks from 82 cattle at Société de Développement et d’Exploitation des Productions Animales (SODEPA) Dumbo ranch (SDR) and 385 ticks from 100 cattle at the Upper Farms ranch (UFR)] by amplification of the open reading frame (ORF) 2 of the pCS20 region of E. ruminantium. PCR amplification of the 279 bp fragment of the pCS20 region detected E. ruminantium DNA in 142 (28.4 %) of the 500 ticks with a higher infection rate (47/115; 40.9 %) observed in ticks from SDR and 24.7 % (95/385) of ticks collected from cattle at UFR. Twenty five randomly selected PCR products were sequenced and results indicated that some of the isolates shared homology with one another and to sequences of E. ruminantium in the GenBank. This report represents the first molecular evidence of E. ruminantium infection in A. variegatum ticks in Cameroon and suggests possible exposure of cattle to this pathogen in our environment.     

Reduced Weight Gain Due to Subclinical Anaplasma phagocytophilum (Formerly Ehrlichia phagocytophila) Infection

Tick-borne fever (TBF) is caused by the rickettsia Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) and is a common disease in sheep in areas of Norway infested by Ixodes ricinus ticks. TBF can cause both direct and indirect losses to sheep kept on tick-infested pastures. In the present work we studied a sheep flock of 26 ewes and 50 lambs on pasture from May until September. No cases of TBF had earlier been observed on this pasture. Blood samples from lambs with a reduced weekly weight gain were collected and analysed for A. phagocytophilum infection by blood smear examination. In addition, at the end of the study, sera from all lambs were analysed by an indirect fluorescent antibody assay (IFA) to determine the antibody titre to E. equi. No clinical signs of tick-borne infections were observed, except in one lamb. However, 30 (60%) of the lambs grazing on this pasture became infected with A. phagocytophilum, and the infected lambs had a reduced weight gain (mean) of 3.8 kg compared with the uninfected lambs. The present study indicates that A. phagocytophilum infection may be widespread and contribute to considerable productivity losses even on pastures with no apparent tick infestation. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Review of Studies on the Transmission of Anaplasma phagocytophilum from Sheep: Implications for the Force of Infection in Endemic Cycles

We review the findings of a longitudinal study of transmission of the intracellular tick-borne bacterium Anaplasma phagocytophilum from sheep to Ixodes ricinus ticks under natural conditions of tick attachment in the UK. In this study, sheep-to-tick transmission efficiency varied in a quadratic relationship with the number of adult ticks that were feeding on the sheep. We raise the hypothesis that this relationship may be due to conflicting effects of the density of ticks on bacterial survival and target cell (neutrophil) fluxes at the tick-host interface: in the same sheep at the same time, resistance to ticks was progressively inhibited with increasing number of feeding adult ticks, and investigation of serological responses to tick antigens suggesting loss of resistance may be associated with polarisation of host Th1 to Th2 type responses to ticks. We also raise the hypothesis that these properties, with superimposed effects on tick survival, may mean that variation in tick density is an important causal factor of observed variations in the force of A. phagocytophilum infection amongst different geographic foci. This revised version was published online in July 2006 with corrections to the Cover Date.     

Arthropod cell lines in the isolation and propagation of tickborne spiroplasmas

The ability of several continuous tick cell culture lines to support growth of tickborne spiroplasmas (helical, wall-less prokaryotes in the classMollicutes) was assessed. Seven triturates, prepared from pools ofIxodes pacificus ticks naturally infected with theSpiroplasma sp. (group VI) organism, were retrieved from frozen (–70°C) storage and passaged in three distinct tick cell lines, in antibiotic-free tick cell culture medium alone, or in spiroplasma culture medium (SP-4 formulation). Six spiroplasma strains were recovered in the RML-19 cell line fromDermacentor variabilis, and five isolations were made in another cell line (RML-15) from this tick species. None was recovered in aRhipicephalus sanguineus cell line (RML-23), in tick cell culture medium, or in SP-4 broth medium. One of the spiroplasma isolates (Y43) was maintained through four consecutive weekly refeedings of theD. variabilis cell line and for three feedings ofR. sanguineus cells, where numbers of spiroplasmas in cell supernatants reached levels comparable to those obtained in the SP-4 medium.A laboratory-adapted strain (SMCA) ofSpiroplasma mirum, a second helical mollicute of tick origin (the suckling mouse cataract agent), grew in three tick cell lines (RML-15, RML-23, and RML-16 cells fromD. parumapertus), in three mosquito cell lines (fromAedes albopictus, Ae. aegypti, andCulex quinquefasciatus), and in both cell culture medium alone and in SP-4 medium. The organisms survived for 1–2 weeks, but failed to multiply, in cell lines fromC. tritaeniorhynchus, Antheraea eucalypti, orXenopus laevis. Some evidence of cytopathic effect ofS. mirum on tick cell lines was seen, although growth of the organism in mosquito cell cultures was not associated with cell toxicity. The use of arthropod cell lines appears to have value in the primary isolation of arthropod- or insect-derived mollicutes and for the study of cytopathogenicity of these wall-less prokaryotes.     

Molecular analysis of tick-borne protozoan and rickettsial pathogens in small ruminants from two South African provinces

Tick-borne protozoan and rickettsial diseases are a major threat to livestock in tropical and sub-tropical regions of Africa. In this study we investigated the presence and distribution of Theileria spp., Babesia ovis, Anaplasma ovis, Anaplasma phagocytophilum, Ehrlichia ruminantium and SFG Rickettsia in sheep and goats from Free State and KwaZulu-Natal provinces. A total of 91 blood samples were screened in this study, 61 from goats and 30 from sheep. PCR assay was conducted using primers based on Theileria spp. 18S rRNA, Babesia ovis (BoSSU rRNA), Anaplasma ovis (AoMSP4), Anaplasma phagocytophilum epank1, Ehrlichia ruminantium pCS20 and SFG Rickettsia OmpA. Overall infection rates of Theileria spp., Anaplasma ovis and Ehrlichia ruminantium were 18 (19.8%), 33 (36.3%) and 13 (14.3%), respectively. The co-infection of two pathogens were detected in 17/91 (18.7%) of all samples, goats having higher rates of co-infection compared to sheep. Phylogenetic tree analysis sequence of pCS20 gene of E. ruminantium of this study was found to be in the same clade with Kumm2 and Riverside strains both from South Africa. The phylogram of SSU rRNA of Theileria ovis had longer branch length compared to all other sequences most of which were from Asia and Middle East. This study provides important data for understanding the tick-borne diseases occurrence in the study area and it is expected to improve the approach for the diagnosis and control of these diseases.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Interspecific hybridizations among species of theElymus semicostatus andElymus tibeticus groups (Poaceae)

Interspecific hybridizations were made between species of theE. semicostatus group, viz.,E. semicostatus (Nees exSteud.)Meld.,E. validus (Meld.)B. Salomon,E. abolinii (Drob.)Tzvel., andE. fedtschenkoi Tzvel., and species of theE. tibeticus group, viz.,E. pendulinus (Nevski)Tzvel.,E. tibeticus (Meld.)Singh,E. shandongensis B. Salomon, andE. gmelinii (Ledeb.)Tzvel., as well as among species within theE. tibeticus group. All species are tetraploid (2n = 4x = 28) and possess SY genomes. Meiotic pairing data from 24 hybrids involving 17 interspecific combinations are presented. The average number of chiasmata per cell ranged from 17.91 to 26.20 in hybrids within theE. tibeticus group, compared with 7.26 to 22.04 in hybrids between the two species groups. Despite the extensive collection of cytological data, there was no definite evidence for confirming or disproving the separate existence of the two groups.     

Ticks and tick-borne pathogens in livestock from nomadic herds in the Somali Region,Ethiopia

Between May 2006 and January 2007, blood samples and ticks were randomly collected from 220 nomadic animals from Filtu and Dollo Odo districts, Libaan zone, in the Somali Region of Ethiopia. Overall, 81.5% cattle, 98.2% camels, 53.4% goats and 61.1% sheep were infested by ixodid ticks. Collected ticks (n = 1,036) were identified as Rhipicephalus pulchellus (40.1%), R. pravus (25.8%), Amblyomma gemma (9.4%), Hyalomma rufipes (13.3%), H. truncatum (2.8%), H. impeltatum (1.2%) and H. dromedarii (0.5%); immature stages (6.1%) belonged to the genera Rhipicephalus and Amblyomma. Tick infestation burden was evaluated by the Tick Abundance Score method on 57 animals from Dollo Odo in August 2006, and it was significantly higher in cattle and camels than in small ruminants (p < 0.001). Reverse Line Blot Hybridisation was applied to detect Theileria, Babesia, Ehrlichia and Anaplasma spp. Five out of 50 blood samples from Filtu, four from cattle and, surprisingly, one from a camel, were positive for Theileria mutans and two from cattle for T. velifera. Adult ticks (n = 104) from both districts were tested and A. gemma from cattle were positive to T. velifera (1) and Ehrlichia ruminantium (5 samples). Positive E. ruminantium samples were also tested by PCR targeting pCS20 and 16S rRNA genes and submitted to DNA sequencing. The phylogenetic reconstruction of pCS20 fragment showed the presence of the Somali region sequences in the East-South African group. Our results are the first available on ticks and selected tick-borne diseases from the Somali region of Ethiopia and could be used as preliminary information for planning sustainable control strategies for tick and tick-borne pathogens in the study area and in neighbouring areas with similar socio-ecological features.     

Characterization of rainbow trout cell lines using microsatellite DNA profiling

Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of the attachment rates of males of the ticksAmblyomma hebraeum andA. variegatum to cattle,sheep and rabbits in the absence of aggregation-attachment pheromone

Losses in domestic ruminants caused by heartwater (Cowdria ruminantium infection) in Zimbabwe and Mozambique are greater when the vector isAmblyomma hebraeum than when the vector isA. variegatum. It has been suggested that the epidemiology of the disease may be influenced by the rates at which unfed adults of these two tick species attach to uninfested hosts (i.e. in the absence of the male-produced aggregation-attachment pheromone [AAP]). In this study we confined unfed males ofA. hebraeum andA. variegatum on uninfested cattle, sheep and rabbits and recorded their attachment rates. Males of both species attached more rapidly on cattle than on sheep or rabbits. Males ofA. hebraeum attached more rapidly than males ofA. variegatum on all three host species. The differences in the attachment rates between the two species were much greater on sheep and rabbits than on cattle. The findings suggest that in the absence of AAP, pioneer males of both tick species may attach to cattle, and pioneer males ofA. hebraeum may also attach to sheep. The differences in the attachment rates ofA. hebraeum andA. variegatum provide a possible explanation for observed differences in the epidemiology of heartwater associated with these two vector species.     

An evaluation of strategic and threshold control measures against the Karoo paralysis tick,Ixodes rubicundus (Acari: Ixodidae) in South Africa

Paralysis caused by feeding female Ixodes rubicundus ticks is a major problem in large areas of South Africa. As the life cycle of the tick extends over a period of 2 years, it was hypothesized that strategic treatment of sheep with an acaricide over a 2 year period, timed to kill most engorging females, should markedly lower the biotic potential of the tick. Two flocks of sheep grazing in separate paddocks known to be infested with I. rubicundus were treated either strategically or on a threshold basis (i.e. only when tick challenge exceeded a predetermined critical level in terms of paralysis) for a 2 year period. The tick burdens of untreated control sheep running with the two flocks were monitored over a 4 year period and their seasonal dynamics determined. The times at which peak infestations occurred were similar for both flocks of sheep, but significant differences in mean tick burdens between the two flocks were recorded. Tick numbers on sheep in the strategically treated flock did not decrease during the third and fourth years of the trial as was expected. Possible reasons for this were low stocking densities, especially during times of peak abundance of adults and the presence of wild hosts which maintained tick populations.     

Presence of growth factors for Methanobacterium ruminantium in both gram-positive and gram-negative bacteria

Autoclaved cells of gram-positive bacteria or mixed rumen organisms promote the growth of rumen strains of Methanobacterium ruminantium, but cells of E. coli were only stimulatory to growth after treatment with lysozyme plus EDTA or with EDTA alone.N-acetylglucosamine is identified as one of the growth factors for rumen strains of Mb. ruminantium.     

Experimental infection of the rabbit tick,<Emphasis Type="Italic"> Haemaphysalis leporispalustris</Emphasis>, with the bacterium<Emphasis Type="Italic"> Rickettsia rickettsii</Emphasis>, and comparative biology of infected and uninfected tick lineages

The present study consisted of two experiments that evaluated experimental infections of Haemaphysalis leporispalustris ticks by a Brazilian strain of Rickettsia rickettsii, and their effect on tick biology. In experiment I, ticks were exposed to R. rickettsii during the larval, nymphal or adult stages by feeding on rabbits (Oryctolagus cuniculus) needle-inoculated with R. rickettsii, and thereafter reared on uninfected rabbits for the entire next tick generation. Regardless of the tick stage that acquired the infection, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 1.3 to 41.7%), and were able to transmit R. rickettsii to uninfected rabbits, as demonstrated by rabbit seroconversion, guinea pig inoculation with rabbit blood, and PCR on rabbit blood. In Experiment II, ticks were exposed to R. rickettsii during the larval stage by feeding on rabbits co-infested with R. rickettsii-infected adult ticks, and thereafter reared on uninfected rabbits until the next generation of larvae. Again, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 3.0 to 40.0%), and were able to transmit R. rickettsii to uninfected rabbits. Thus, it was demonstrated that larvae, nymphs, and adults of H. leporispalustris were able to acquire and maintain the R. rickettsii infection by transstadial and transovarial transmissions within the tick population, with active transmission of the bacterium to susceptible rabbits by all parasitic stages. Analyses of biological parameters of uninfected and R. rickettsii-infected tick lineages were performed in order to evaluate possible deleterious effects of R. rickettsii to the infected tick lineages. Surprisingly, all but one of the four R. rickettsii-experimental groups of the present study showed overall better biological performance than their sibling uninfected control ticks. Results of the present study showed that H. leporispalustris could support infection by a high virulent strain of R. rickettsii for at least two generations, in which infected tick lineages tended to have better performance than uninfected ticks. Our results support a possible role of H. leporispalustris in the enzootic maintenance of R. rickettsii in Latin America, as previously suggested by earlier works.     

Suitability of different media for in vitro cultivation of the ruminal protozoa species Entodinium caudatum, Eudiplodinium maggii, and Epidinium ecaudatum

Three protozoal cultivation media were tested to determine the medium which best facilitated growth and viability of key B-type ciliates isolated from the sheep rumen. Entodinium caudatum and Eudiplodinium maggii were grown anaerobically in 50-ml flasks for 32 days in Caudatum-type (C), Kisidayova (K) or Dehority (M) medium. On day 32, in media K and M, E. caudatum cell counts were high with 5.6 × 10³ and 7.8 × 10³ mL⁻¹, respectively, and the proportion of dead cells was low with 0.6 and 1.4%, respectively. E. maggii concentrations when grown in medium M and C were 2.7 × 10³ and 2.4 × 10³ mL⁻¹, respectively, with 3.9 and 14.1% dead cells. Medium M, which favoured growth of both protozoa species, was tested again and Epidinium ecaudatum was included. Protozoa were grown for a 4-month period and samples were taken in the last two months on days 1, 7, 35 and 57. Average cell concentrations were 10.0, 0.8 and 0.5 × 10³ mL⁻¹ for E. caudatum, E. maggii, and E. ecaudatum, respectively. In conclusion, medium M would appear to be the best choice for cultivating these three species in one medium.     

Analysis of Methanogen Diversity in the Rumen Using Temporal Temperature Gradient Gel Electrophoresis: Identification of Uncultured Methanogens

A temporal temperature gradient gel electrophoresis (TTGE) method was developed to determine the diversity of methanogen populations in the rumen. Tests with amplicons from genomic DNA from 12 cultured methanogens showed single bands for all strains, with only two showing apparently comigrating bands. Fingerprints of methanogen populations were analyzed from DNA extracted from rumen contents from two cattle and four sheep grazing pasture. For one sheep, dilution cultures selective for methanogens were grown and the culturable methanogens in each successive dilution examined by TTGE. A total of 66 methanogen sequences were retrieved from bands in fingerprints and analyzed to reveal the presence of methanogens belonging to the Methanobacteriales, the Methanosarcinales, and to an uncultured archaeal lineage. Twenty-four sequences were most similar to Methanobrevibacter ruminantium, five to Methanobrevibacter smithii, four to Methanosphaera stadtmanae, and for three, the nearest match was Methanimicrococcus blatticola. The remaining 30 sequences did not cluster with sequences from cultured archaea, but when combined with published novel sequences from clone libraries formed a monophyletic lineage within the Euryarchaeota, which contained two previously unrecognized clusters. The TTGE bands from this lineage showed that the uncultured methanogens had significant population densities in each of the six rumen samples examined. In cultures of dilutions from one rumen sample, TTGE examination revealed these methanogens at a level of at least 10⁵ g⁻¹. Band intensities from low-dilution cultures indicated that these methanogens were present at similar densities to Methanobrevibacter ruminantium-like methanogens, the sole culturable methanogens in high dilutions (10⁶–10⁻¹⁰ g⁻¹). It is suggested that the uncultured methanogens together with Methanobrevibacter spp. may be the predominant methanogens in the rumen. The TTGE method presented in this article provides a new opportunity for characterizing methanogen populations in the rumen microbial ecosystem.     

Experimental transmission ofCowdria ruminantium (Rickettsiales) by the American reptile tickAmblyomma dissimile Koch, 1844

A Senegalese isolate of the rickettsiaCowdria ruminantium was transmitted transstadially by nymphs of the American reptile tickAmblyomma dissimile. Only eight nymphs, fed as larvae on a Saanen goat reacting to heartwater, were required to transmit fatal heartwater to another susceptible goat.SinceA. dissimile usually feeds on snakes, iguanas and lizards in central America, the tick is not considered to play a significant role in the transmission of heartwater between ruminants. However, the tick could play a role in maintaining a rickettsial reservoir in reptile populations, since it has been shown that an African reptile can be a subclinical carrier ofC. ruminantium, infective to vector ticks.     

Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) and Iberian red deer (<Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis>) in central Spain

Emerging tick-borne diseases of humans and animals have occurred frequently during the past 30 years. These disease outbreaks appear to result from changes in the distribution of tick and vertebrate hosts, and the introduction of humans and domestic animals into tick–pathogen–wildlife cycles. Use of molecular technologies now available for identification of pathogens in ticks can provide valuable information that allows for risk analysis of emerging tick-borne diseases. In this study, the prevalence of selected pathogens in ticks collected in six locations in central Spain from the major wild ungulate species, European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus), was determined by PCR. Tick species collected included Ixodes ricinus, Dermacentor marginatus, Rhipicephalus bursa and Hyalomma m. marginatum. Pathogens identified in ticks included piroplasmids, Anaplasma spp., Ehrlichia spp. and Rickettsia spp. Piroplasmids were identified in all tick species except I. ricinus. Ehrlichia spp. were detected in all tick species and collection locations, while Rickettsia spp., which proved to be R. slovaca and a recently identified Rickettsia sp. DnS28, were identified only in D. marginatus. A. marginale and A. phagocytophilum were detected in D. marginatus, R. bursa and Hy. m. marginatum. Concurrent infections of these pathogens were frequently observed in ticks. Notably, A. phagocytophilum, which is infective for a broad host range that includes humans and domestic and wild animals, was identified in ticks from all collection locations. The variety of ticks and tick-borne pathogens demonstrated in this study suggests a risk in central Spain for the emergence of tick-borne diseases in humans and domestic animals.     

Factors affecting the distributions of the ticks Amblyomma hebraeum and A. variegatum in Zimbabwe: implications of reduced acaricide usage

The ticks Amblyomma hebraeum and A. variegatum are the main vectors of heartwater, a disease of ruminants caused by Cowdria ruminantium, in the agricultural areas of Zimbabwe. At present, A. hebraeum is widely distributed in the dry southern lowveld, and occurs in at least seven foci in the higher rainfall highveld. Amblyomma variegatum occurs in the Zambezi valley and surrounding dry lowveld areas in the northwest. The distribution of A. hebraeum has changed considerably over the past 70 years, while that of A. variegatum appears to have remained fairly static. The distribution patterns of both species in Zimbabwe display anomalous features; the ticks occur in areas of lowest predicted climatic suitability for survival and development and in areas where the densities of cattle, the most important domestic host, are lowest. The only factor favouring the survival of the species in the lowveld habitats in which they occur is the presence of alternative wildlife hosts for the adult stage. Their absence from more climatically favourable highveld habitats appears to have been the result of intensive acaricide treatment of cattle over a long period and a historic absence of significant numbers of wildlife hosts. Eradication of A. hebraeum and A. variegatum by intensive acaricide treatment of cattle can be achieved in the absence of significant numbers of alternative hosts, because of the long attachment and feeding periods of the adults of these tick species. However, eradication becomes impossible when alternative hosts for the adult stage are present, because a pheromone emitted by attached males attracts the unfed nymphal and adult stages to infested hosts. The unfed ticks are not attracted to uninfested hosts, such as acaricide-treated cattle.Regular acaricide treatment of cattle is expensive and so, for economic reasons, the Government of Zimbabwe is no longer enforcing a policy of strict tick control. It is likely that reduced tick control will result in the spread of Amblyomma ticks to previously uninfested areas. Added to this, recent introductions of various wildlife species to highveld commercial farming areas have created conditions in which the ticks could become established in higher rainfall areas. Amblyomma hebraeum is more likely to spread than A. variegatum, because its adults parasitize a wider range of wildlife hosts (warthogs, medium to large-sized antelope, giraffe, buffalo and rhinoceros), whereas adults of A. variegatum appear to be largely restricted to one wildlife species (buffalo) in Zimbabwe, the distribution of which is now confined to very limited areas of the country, as part of foot and mouth disease control measures. A model to predict the rate of spread of A. hebraeum through the highveld is described.Possible control options for dealing with the spread of Amblyomma ticks and heartwater to previous unaffected highveld areas, include (1) continuation of intensive acaricide treatment of cattle to prevent the spread, (2) establishment of a buffer zone of intensive tick control around affected areas to contain the spread and (3) allow the spread to occur and control heartwater by means of immunization. An economic analysis to determine the costs and benefits of the control options, which takes into account the development of Amblyomma-specific tick control technologies and improved heartwater vaccines, is recommended.Deceased.