Molecular detection of tick-borne protozoan parasites in sika deer (Cervus nippon) from western regions of Japan

The sika deer (Cervus nippon) is one of the most common species of wildlife in Japan. This study aimed to reveal the prevalence of tick-borne protozoan parasites in wild sika deer living in western Japan. We used nested polymerase chain reaction (PCR) to detect the 18S rRNA gene of tick-borne apicomplexan parasites (Babesia, Theileria, and Hepatozoon spp.) from 276 blood and liver samples from sika deer captured in the Yamaguchi, Oita, Kagoshima, Okayama, Ehime, Kochi, and Tokushima Prefectures. In total, 259 samples (259/276; 93.8%) tested positive in the nested PCR screening. Gene sequencing revealed that 99.6% (258/259) of positive samples contained Theileria sp. (sika 1), while Theileria sp. (sika 2), another Theileria species, was detected in only 3 samples. We also found that one sample from a sika deer captured in Kagoshima contained the gene of an unidentified Babesia sp. related to Babesia sp. Kh-Hj42, which was previously collected from tick in western Siberia. In conclusion, we found a high prevalence of piroplasms in sika deer from western Japan, and DNA analysis revealed that Theileria sp. (sika 1) had the highest infection rate.     

Prevalence and phylogenetic analysis of Cryptosporidium infections in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido,Japan

The Yezo sika deer (Cervus nippon yesoensis) on the island of Hokkaido, Japan are currently recognized as overabundant. Hunting is used to control the deer population, and this has increased the supply of game meat, which is associated with a high risk of various food-borne infections. Additionally, the sub-prefecture Tokachi has a dense population of livestock, which are potentially at risk of cross-species infections from the deer. In this study, we undertook the first analysis of the incidence of Cryptosporidium infection in the Yezo sika deer in the Tokachi area using polymerase chain reaction testing and phylogenetic analysis. Polymerase chain reaction analysis showed Cryptosporidium species present in 7.5% of fecal samples (13/173) collected from deer hunted between 2016 and 2017. However, the zoonotic Cryptosporidium paruvm parasite was not detected in the phylogenetic analysis; when sequenced, all species in the positive samples matched the Cryptosporidium deer genotype. However, deer may act as a reservoir of the zoonotic Cryptosporidium parvum parasite, which affects both humans and livestock. Therefore, we recommend the continuation of surveys of the incidence of Cryptosporidium infections in Yezo sika deer.     

Molecular epidemiology of bovine Babesia spp. and Theileria orientalis parasites in beef cattle from northern and northeastern Thailand

Beef cattle production represents the largest cattle population in Thailand. Their productivity is constrained by tick-borne diseases such as babesiosis and theileriosis. In this study, we determined the prevalence of Babesia bigemina, Babesia bovis and Theileria orientalis using polymerase chain reaction (PCR). The genetic markers that were used for detection of the above parasites were sequenced to determine identities and similarity for Babesia spp. and genetic diversity of T. orientalis. Furthermore the risk factors for the occurrence of the above protozoan parasites in beef cattle from northern and northeastern parts of Thailand were assessed. A total of 329 blood samples were collected from beef cattle in 6 provinces. The study revealed that T. orientalis was the most prevalent (30.1%) parasite in beef cattle followed by B. bigemina (13.1%) and B. bovis (5.5%). Overall, 78.7% of the cattle screened were infected with at least one of the above parasites. Co-infection with Babesia spp. and T. orientalis was 30.1%. B. bigemina and T. orientalis were the most prevalent (15.1%) co-infection although triple infection with the three parasites was observed in 3.0% of the samples. Sequencing analysis revealed that B. bigemina RAP1 gene and B. bovis SBP2 gene were conserved among the parasites from different cattle samples. Phylogenetic analysis showed that the T. orientalis MPSP gene from parasites isolated from cattle in north and northeast Thailand was classified into types 5 and 7 as reported previously. Lack of tick control program was the universal risk factor of the occurrence of Babesia spp. and T. orientalis infection in beef cattle in northern and northeastern Thailand. We therefore recommend training of farmers on appropriate tick control strategies and further research on potential vectors for T. orientalis and elucidate the effect of co-infection with Babesia spp. on the pathogenicity of T. orientalis infection on beef in northern and northeastern Thailand.     

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture,Japan revealed by ELISA using recombinant cathepsin L1

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.     

First survey of ticks,tick-borne pathogens (Theileria,Babesia, Anaplasma and Ehrlichia) and Trypanosoma evansi in protected areas for threatened wild ruminants in Tunisia

The aim of the study was to identify ticks present in the environment and wild Tunisian ruminants and to detect tick-borne pathogens and Trypanosoma evansi DNA in these specimens. Sampling was done throughout each season from the environment in three protected areas around Tunisia: El Feidja, Haddaj and Oued Dekouk. Ticks were collected also, from one fawn of Barbary red deer and eight naturally deceased wild ruminants (one Barbary red deer, five Scimitar-horned oryx, one Addax antelope and one Dorcas gazelle), all of which lived in various protected areas. PCR and nested PCRs were performed to detect the presence of Theileria spp., Babesia spp., Trypanosoma evansi, Ehrlichia spp., Anaplasma spp., Anaplasma bovis and Anaplasma phagocytophilum DNA in these tick specimens. A total of 352 ticks were collected, belonging to six different species: Hyalomma excavatum (80.6%), Hyalomma dromedarii (10.2%), Hyalomma marginatum (0.5%), Rhipicephalus bursa (0.5%), Rhipicephalus sanguineus sensu lato (5.1%) and Ixodes ricinus (2.8%). Pathogens have been detected in 25% of H. dromedarii, 9.1% of H. excavatum and 5% of R. sanguineus sensu lato. The percentage of detection of T. evansi was 0.2%. Ehrlichia spp.-Anaplasma spp. were detected in 10.1% of ticks. Anaplasma spp. and A. bovis were detected in 7.6%, and 0.8% of examined ticks, respectively. None of the Theileria spp., Babesia spp., or A. phagocytophilum DNA was detected in the tested ticks. To our knowledge, the present study represents the first identification of these six tick species and the first detection of rickettsial pathogens and T. evansi in North African wild ruminants' species. These results extend the knowledge about the diversity of ticks and tick-borne pathogens in wildlife and justify further investigations of the possible role of R. sanguineus sensu lato in the transmission of T. evansi.     

Identification of Parasitic Communities within European Ticks Using Next-Generation Sequencing

Background

Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.

Methodology/Principal Findings

We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.

Conclusions/Significance

Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.     

Theileria infection with severe anemia and unhealed fracture in a sika deer Cervus nippon aplodontus (Cervidae: Cetartiodactyla)

An adult female sika deer (Cervus nippon aplodontus) inhabiting Nara Park, Nara, Japan, had broken bone injuries from a car accident. During its treatment, we found that the sika deer had severe anemia and the fracture remained unhealed throughout. Peripheral blood smear revealed piroplasms in the erythrocytes, which were identified as merozoites of undescribed Theileria species, widely found in sika deer in Japan. This is the report of a clinical case of Theileria infection, accompanied by severe anemia in a sika deer.     

Babesia spp. Identified by PCR in Ticks Collected from Domestic and Wild Ruminants in Southern Switzerland

Concurrent infections with vector-borne pathogens affected a cattle herd in Switzerland, and one of the pathogens was identified as Babesia bigemina, which had never been observed in this country before. Therefore, a survey of the occurrence of ruminant Babesia spp. and their tick vectors in Switzerland was conducted. A total of 2,017 ticks were collected from sheep, goats, cattle, and wild ruminants (deer, roe deer, and chamois) in southern parts of Switzerland and identified morphologically. The vast majority of the ticks (99.2%) were Ixodes ricinus, but 14 ticks from sheep and goats were identified as Dermacentor marginatus and two ticks from wild ruminants were identified as Hemaphysalis punctata. PCR analyses of 700 ticks revealed the presence of Babesia divergens (n = 6), Babesia sp. genotype EU1 (n = 14), and B. major (n = 2), whose suggested occurrence was confirmed in this study by molecular analysis, and the presence of novel Babesia sp. genotype CH1 (n = 4), which is closely related to B. odocoilei and to Babesia sp. genotype RD61 reported from North America. The identification of B. divergens and B. major in ticks collected from wild ruminants cast doubt on the postulated strict host specificity of these bovine Babesia species. Furthermore, the zoonotic Babesia sp. genotype EU1 was detected in ticks collected from domestic animals but was obtained predominantly from ticks collected from wild ruminants. More than one tick containing DNA of different Babesia spp. were collected from two red deer. Hence, the role of these game animals as reservoir hosts of Babesia spp. seems to be important but requires further investigation.     

Molecular investigation of tick-borne pathogens in ticks from grazing cattle in Korea

This study was carried out to identify the tick species that infest grazing cattle and to determine the presence of tick-borne pathogens transmitted by these ticks in Korea. A total of 903 ticks (categorized into 566 tick pools) were collected from five provinces during 2010–2011. The most prevalent tick species was Haemaphysalis longicornis, followed by three Ixodes spp. ticks. The collected ticks were infected with both rickettsial and protozoan pathogens. In all, 469 (82.9%) tick pools tested positive for the Anaplasma/Ehrlichia 16S rRNA gene, whereas 67 (11.8%) were positive for the Babesia/Theileria 18S rRNA gene. Among the rickettsial pathogens, E. canis was detected with the highest rate (22.3%), followed by A. platys (20%), E. chaffeensis (19.4%), E. ewingii (19.3%), Rickettsia sp. (12.4%), A. phagocytophilum (5.5%) and E. muris (0.5%). Among the protozoan pathogens, T. equi was detected with the highest rate (7.2%), followed by T. sergenti/T. buffeli (3.7%) and B. caballi (0.35%). Simultaneous infections with up to seven pathogens were also identified. In particular, ticks infected with rickettsial pathogens were also infected with protozoan pathogens (22 samples). All five provinces investigated infected with tick-borne pathogens.     

Tick burden on European roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

In our study we assessed the tick burden on roe deer (Capreolus capreolus L.) in relation to age, physical condition, sex, deer density and season. The main objective was to find predictive parameters for tick burden. In September 2007, May, July, and September 2008, and in May and July 2009 we collected ticks on 142 culled roe deer from nine forest departments in Southern Hesse, Germany. To correlate tick burden and deer density we estimated deer density using line transect sampling that accounts for different detectability in March 2008 and 2009, respectively. We collected more than 8,600 ticks from roe deer heads and necks, 92.6% of which were Ixodes spp., 7.4% Dermacentor spp. Among Ixodes, 3.3% were larvae, 50.5% nymphs, 34.8% females and 11.4% males, with significant seasonal deviation. Total tick infestation was high, with considerable individual variation (from 0 to 270 ticks/deer). Adult tick burden was positively correlated with roe deer body indices (body mass, age, hind foot length). Significantly more nymphs were found on deer from forest departments with high roe deer density indices, indicating a positive correlation with deer abundance. Overall, tick burden was highly variable. Seasonality and large scale spatial characteristics appeared to be the most important factors affecting tick burden on roe deer.     

The role of deer as vehicles to move ticks, Ixodes ricinus, between contrasting habitats

In Europe the most important hosts maintaining Ixodes ricinus tick populations are deer. Therefore, excluding deer by fencing or culling are potential tick management tools. Here we test the hypothesis that deer act as vehicles for moving ticks between two distinct habitats: forest and open heather moorland. We utilised an ideal “natural experiment” whereby forests were either fenced or unfenced to prevent or allow deer to move between habitats. We aimed to test the hypothesis that deer cause a net movement of ticks from high tick density areas, i.e. forests, to low tick density areas, i.e. open moorland. We recorded I. ricinus and host abundance in 10 unfenced and seven fenced forests and their respective surrounding heather moorland. We found that fenced forests had fewer deer and fewer I. ricinus nymphs than unfenced forests. However, we found no evidence that fencing forests reduced I. ricinus abundance on adjacent heather moorland. Thus there was insufficient evidence for our hypothesis that deer cause a net movement of ticks from forest onto adjacent moorland. However, we found that deer abundance generally correlates with I. ricinus abundance. We conclude that fencing can be used as a tool to reduce ticks and disease risk in forests, but that fencing forests is unlikely to reduce ticks or disease risk on adjacent moorland. Instead, reducing deer numbers could be a potential tool to reduce tick abundance with implications for disease mitigation.     

Simultaneous detection of piroplasma infections in field Haemaphysalis qinghaiensis ticks by reverse line blotting

Piroplasmosis is caused by tick-borne haemoprotozoan parasites in the genera of Theileria and Babesia, in which numbers of agents are highly pathogenic for cattle, sheep and goats. We developed a reverse line blot (RLB) assay for detection and differentiation of four different parasites, in which 18S ribosomal RNA gene sequence was amplified with a set of universal primers specific for all members in the genera of Theileria and Babesia; and the probes were designed on the basis of hypervariable region 4 (V4 region) of 18S rRNA gene. Three Theileria and one Babesia can be detected simultaneously on this system and it was sensitive to detect a parasitemia level between 10⁻⁵ and 10⁻⁸%. A total of 149 Haemaphysalis qinghaiensis ticks collected from Lintan County of Gannan Tibetan Autonomous Region was tested by RLB. Among these, 136 tick samples were also tested by a nested PCR assay developed previously. After comparison of these results, it showed that more T. luwenshuni was detected in RLB assay, while more T. uilenbergi was detected in H. qinghaiensis ticks by nested PCR. The RLB has shown capability for simultaneous detection of four species of piroplasm in H. qinghaiensis ticks, indicating its usefulness for epidemiological studies of piroplasmosis.     

Influence of the Biotope on the Tick Infestation of Cattle and on the Tick-Borne Pathogen Repertoire of Cattle Ticks in Ethiopia

Background

The majority of vector-borne infections occur in the tropics, including Africa, but molecular eco-epidemiological studies are seldom reported from these regions. In particular, most previously published data on ticks in Ethiopia focus on species distribution, and only a few molecular studies on the occurrence of tick-borne pathogens or on ecological factors influencing these. The present study was undertaken to evaluate, if ticks collected from cattle in different Ethiopian biotopes harbour (had access to) different pathogens.

Methods

In South-Western Ethiopia 1032 hard ticks were removed from cattle grazing in three kinds of tick biotopes. DNA was individually extracted from one specimen of both sexes of each tick species per cattle. These samples were molecularly analysed for the presence of tick-borne pathogens.

Results

Amblyomma variegatum was significantly more abundant on mid highland, than on moist highland. Rhipicephalus decoloratus was absent from savannah lowland, where virtually only A. cohaerens was found. In the ticks Coxiella burnetii had the highest prevalence on savannah lowland. PCR positivity to Theileria spp. did not appear to depend on the biotope, but some genotypes were unique to certain tick species. Significantly more A. variegatum specimens were rickettsia-positive, than those of other tick species. The presence of rickettsiae (R. africae) appeared to be associated with mid highland in case of A. variegatum and A. cohaerens. The low level of haemoplasma positivity seemed to be equally distributed among the tick species, but was restricted to one biotope type.

Conclusions

The tick biotope, in which cattle are grazed, will influence not only the tick burden of these hosts, but also the spectrum of pathogens in their ticks. Thus, the presence of pathogens with alternative (non-tick-borne) transmission routes, with transstadial or with transovarial transmission by ticks appeared to be associated with the biotope type, with the tick species, or both, respectively.     

Massive Infection of Seabird Ticks with Bacterial Species Related to Coxiella burnetii

Seabird ticks are known reservoirs of bacterial pathogens of medical importance; however, ticks parasitizing tropical seabirds have received less attention than their counterparts from temperate and subpolar regions. Recently, Rickettsia africae was described to infect seabird ticks of the western Indian Ocean and New Caledonia, constituting the only available data on bacterial pathogens associated with tropical seabird tick species. Here, we combined a pyrosequencing-based approach with a classical molecular analysis targeting bacteria of potential medical importance in order to describe the bacterial community in two tropical seabird ticks, Amblyomma loculosum and Carios (Ornithodoros) capensis. We also investigated the patterns of prevalence and host specificity within the biogeographical context of the western Indian Ocean islands. The bacterial community of the two tick species was characterized by a strong dominance of Coxiella and Rickettsia. Our data support a strict Coxiella-host tick specificity, a pattern resembling the one found for Rickettsia spp. in the same two seabird tick species. Both the high prevalence and stringent host tick specificity suggest that these bacteria may be tick symbionts with probable vertical transmission. Detailed studies of the pathogenicity of these bacteria will now be required to determine whether horizontal transmission can occur and to clarify their status as potential human pathogens. More generally, our results show that the combination of next generation sequencing with targeted detection/genotyping approaches proves to be efficient in poorly investigated fields where research can be considered to be starting from scratch.     

Ehrlichia chaffeensis Infection in the Reservoir Host (White-Tailed Deer) and in an Incidental Host (Dog) Is Impacted by Its Prior Growth in Macrophage and Tick Cell Environments

Ehrlichia chaffeensis, transmitted from Amblyomma americanum ticks, causes human monocytic ehrlichiosis. It also infects white-tailed deer, dogs and several other vertebrates. Deer are its reservoir hosts, while humans and dogs are incidental hosts. E. chaffeensis protein expression is influenced by its growth in macrophages and tick cells. We report here infection progression in deer or dogs infected intravenously with macrophage- or tick cell-grown E. chaffeensis or by tick transmission in deer. Deer and dogs developed mild fever and persistent rickettsemia; the infection was detected more frequently in the blood of infected animals with macrophage inoculum compared to tick cell inoculum or tick transmission. Tick cell inoculum and tick transmission caused a drop in tick infection acquisition rates compared to infection rates in ticks fed on deer receiving macrophage inoculum. Independent of deer or dogs, IgG antibody response was higher in animals receiving macrophage inoculum against macrophage-derived Ehrlichia antigens, while it was significantly lower in the same animals against tick cell-derived Ehrlichia antigens. Deer infected with tick cell inoculum and tick transmission caused a higher antibody response to tick cell cultured bacterial antigens compared to the antibody response for macrophage cultured antigens for the same animals. The data demonstrate that the host cell-specific E. chaffeensis protein expression influences rickettsemia in a host and its acquisition by ticks. The data also reveal that tick cell-derived inoculum is similar to tick transmission with reduced rickettsemia, IgG response and tick acquisition of E. chaffeensis.     

Evaluation of a Real-Time PCR Test for the Detection and Discrimination of Theileria Species in the African Buffalo (Syncerus caffer)

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected.Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.     

The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China

The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 μl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.     

Tick community composition in Midwestern US habitats in relation to sampling method and environmental conditions

The ranges of many tick species are changing due to climate change and human alteration of the landscape. Understanding tick responses to environmental conditions and how sampling method influences measurement of tick communities will improve our assessment of human disease risk. We compared tick sampling by three collection methods (dragging, CO₂ trapping and rodent surveys) in adjacent forested and grassland habitats in the lower Midwest, USA, and analyzed the relationship between tick abundance and microclimate conditions. The study areas were within the overlapping ranges of three tick species, which may provide conditions for pathogen exchange and spread into new vectors. Dermacentor variabilis (American dog tick) was found using all methods, Amblyomma americanum (lonestar tick) was found by dragging and CO₂ trapping and Ixodes scapularis (blacklegged deer tick) was found only on rodents. Proportion of each species differed significantly among sampling methods. More ticks were found in forests compared to open habitats. Further, more ticks were collected by dragging and from rodents in hotter, drier conditions. Our results demonstrate that multiple sampling methodologies better measure the tick community and that microclimate conditions strongly influence the abundance and activity of individual tick species.     

Failure of the Amblyomma cajennense nymph to become infected by Theileria equi after feeding on acute or chronically infected horses

Tick-borne diseases in horses are caused by the intraerythrocytic protozoan parasites Theileria equi and Babesia caballi. Although T. equi is highly endemic in Latin America, the New World vector of this important parasite is controversial. The aim of this study was to test the ability of nymph Amblyomma cajennense ticks acquire infection by T. equi following feeding on infected horses. Three experiments were performed: tick acquisition of T. equi from an experimentally infected horse, tick acquisition of T. equi from naturally infected foals and tick acquisition of T. equi from a chronically infected horse. A. cajennense adults were dissected and salivary glands were collected in aliquots. Methyl green pyronin staining of the salivary glands did not show the presence of hypertrophy of acini or cell nuclei normally suggestive of Theileria spp. infection. The pools of salivary glands were negative for Theileria DNA in nested PCR assays. Histopathological analysis failed to detect sporoblast and sporozoites of T. equi in salivary gland acini. This study was not able to observe infection of the A. cajennense by T. equi.     

Molecular identification of tick-borne pathogens in ticks collected from dogs and small ruminants from Greece

Ticks are vectors for a variety of human and animal pathogens (bacteria, protozoa and viruses). In order to investigate the pathogens carried by ticks in Greece, a total of 179 adult ticks (114 female and 65 male) were collected from domestic animals (sheep, goats and dogs) from 14 prefectures of six regions of Greece. Among them, 40 were Dermacentor marginatus, 25 Haemaphysalis parva, 22 H. sulcata, one H. punctata, 13 Ixodes gibbosus, 77 Rhipicephalus sanguineus s.l. and one R. bursa. All ticks were tested for the presence of DNA of Anaplasma spp., Babesia spp., Coxiella burnetii, Rickettsia spp. and Theileria spp. The collected ticks were examined by PCR and reverse line blot (RLB) assay. A prevalence of 20.1% for Anaplasma spp., 15.6% for Babesia spp. (identifying B. bigemina, B. divergens, B. ovis and B. crassa), 17.9% for C. burnetii, 15.1% for Rickettsia spp., and 21.2% for Theileria spp. (identifying T. annulata, T. buffeli/orientalis, T. ovis and T. lestoquardi) was found. The results of this study demonstrate the variety of tick-borne pathogens of animal and human importance circulating in Greece, and that awareness is needed to minimize the risk of infection, especially among farmers and pet owners.