Rhipicephalus Appendiculatus Research Articles

The Emergence of Theileria parva in Jonglei State, South Sudan: Confirmation Using Molecular and Serological Diagnostic Tools.

A cross-sectional survey was carried out in four counties of Jonglei State, South Sudan, between May and June 2012 to determine the distribution and northern limit of Theileria parva, the causative agent of East Coast fever in cattle, and its tick vector Rhipicephalus appendiculatus, as a prerequisite to the deployment of relevant control strategies. A total of 1636 ticks, 386 serum samples and 399 blood samples were collected from indigenous, apparently healthy, cattle of different agegroups. Tick species were identified morphologically, and the identity of R.appendiculatus was confirmed by DNA barcoding. Overall, the T.parva infection rate in R.appendiculatus was 25% as shown by nested PCR. ELISA was used to assess antibodies to T.parva, and the overall seroprevalence was 22.8%. PCR of the blood samples showed 55 (13.8%) were positive for T.parva. This is the first molecular confirmation of T.parva DNA in areas north of Juba, where it was previously known and established. The northern limit of T.parva was determined as N⁰06.17.792, about 242Km north from Juba. Implication of this limit on the epidemiology and control of ECF is discussed.

Extensive polymorphism of Ra86 genes in field populations of Rhipicephalus appendiculatus from Kenya.

Commercial vaccines based on recombinant forms of the Bm86 tick gut antigen are used to control the southern cattle tick, Rhipicephalus microplus, a 1-host species, in Australia and Latin America. We describe herein sequence polymorphism in genes encoding Ra86 homologues of Bm86 in the brown ear tick, Rhipicephalus appendiculatus, isolated from four Kenyan field populations and one laboratory colony. Sequencing of 19 Ra86 sequences defined two alleles differentiated by indels, encoding 693 amino acids (aa) and 654 aa respectively, from the Muguga laboratory reference strain. Ra86 sequences were also determined from gut cDNA from four field populations of R. appendiculatus collected in different livestock production systems in Kenya. Analysis of approximately 20 Ra86 sequences from each of the four field sites in central and Western Kenya; Makuyu, Kiambu, Kakamega and Uasin Gishu, revealed three additional size types differentiated by 39–49 amino acid indels resulting in a total of 5 indel-defined genotypes. The 693 aa type 5 was isolated only from the laboratory tick stock; genotypes 1, 2 and 3 were identified in ticks from the four Kenyan field sites and appeared to be derivatives of the shorter RA86 genotype found in Muguga laboratory stock genotype 4. By contrast no large indels have yet been observed between R. microplus sequences from Australia, South America or Africa.Evidence that selection contributes to the observed sequence variation was provided by analysis of ratio of synonymous and non-synonymous substitutions and application of the selective neutrality and neutral evolution tests to the primary data. Phylogenetic analysis clustered sequences from all Ra86 size types and Bm86, into four major clades based on amino acid substitutions, but there was no evidence that these groupings correlated with geographical separation of R. appendiculatus populations

De novo assembly and annotation of the salivary gland transcriptome of Rhipicephalus appendiculatus male and female ticks during blood feeding

Tick secretory proteins modulate haemostasis, inflammation and immune responses of the host and are attractive recombinant anti-tick vaccine candidates. Yet, many of the proteins have not been characterised due to the limited sequence availability for ticks and other arthropods for homology-based annotation. To address this limitation, we sequenced the salivary glands of the economically important adult male and female Rhipicephalus appendiculatus ticks during feeding. The quality-filtered Illumina sequencing reads were de novo assembled to generate a R. appendiculatus sialotranscriptome of 21,410 transcripts. A non-redundant set of 12,761 R. appendiculatus proteins was predicted from the transcripts, including 2134 putative secretory and 8237 putative housekeeping proteins. Secretory proteins accounted for most of the expression in the salivary gland transcriptome (63%). Of the secretory protein class, the Glycine-rich superfamily contributed 66% and the Lipocalin family 12% of the transcriptome expression. Differential expression analysis identified 1758 female and 2346 male up-regulated transcripts, suggesting varying blood-feeding mechanisms employed between female and male ticks. The sialotranscriptome assembled in this work, greatly improves on the sequence information available for R. appendiculatus and is a valuable resource for potential future vaccine candidate selection.

Emergence of multi-acaricide resistant Rhipicephalus ticks and its implication on chemical tick control in Uganda.

BackgroundAcaricide failure has been on the rise in the western and central cattle corridor of Uganda. In this study, we identified the tick species associated with acaricide failure and determined their susceptibility to various acaricide molecules used for tick control in Uganda.MethodsIn this cross sectional study, tick samples were collected and identified to species level from 54 purposively selected farms (from 17 districts) that mostly had a history of acaricide failure. Larval packet test was used to screen 31 tick populations from 30 farms for susceptibility at discriminating dose (DD) and 2 × DD of five panels of commercial acaricide molecules belonging to the following classes; amidine, synthetic pyrethroid (SP), organophosphate (OP) and OP-SP co-formulations (COF). Resistance was assessed based on World Health Organization criteria for screening insecticide resistance.ResultsOf the 1357 ticks identified, Rhipicephalus (Rhipicephalus) appendiculatus and Rhipicephalus (Boophilus) decoloratus were the major (95.6 %) tick species in farms sampled. Resistance against SP was detected in 90.0 % (27/30) of the tick populations tested. Worryingly, 60.0 % (18/30) and 63.0 % (19/30) of the above ticks were super resistant (0 % mortality) against 2 × DD cypermethrin and deltamethrin, respectively. Resistance was also detected against COF (43.3 %), OP chlorfenvinphos (13.3 %) and amitraz (12.9 %). In two years, 74.1 % (20/27) of the farms had used two to three acaricide molecules, and 55.6 % (15/27) rotated the molecules wrongly. Multi-acaricide resistance (at least 2 molecules) was detected in 55.2 % (16/29) of the resistant Rhipicephalus ticks and significantly associated with R. decoloratus (p = 0.0133), use of both SP and COF in the last 2 years (p < 0.001) and Kiruhura district (p = 0.0339). Despite emergence of amitraz resistance in the greater Bushenyi area, it was the most efficacious molecule against SP and COF resistant ticks.ConclusionThis study is the first to report emergence of super SP resistant and multi-acaricide resistant Rhipicephalus ticks in Uganda. Amitraz was the best acaricide against SP and COF resistant ticks. However, in the absence of technical interventions, farmer-led solutions aimed at troubleshooting for efficacy of multitude of acaricides at their disposal are expected to potentially cause negative collateral effects on future chemical tick control options, animal welfare and public health.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1278-3) contains supplementary material, which is available to authorized users.

Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines.

This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

Vasoconstriction induced by salivary gland extracts from ixodid ticks

In their quest for blood, most haematophagous parasites secrete vasodilators in their saliva to counter the host haemostatic response of vasoconstriction. Surprisingly, salivary gland extracts from adult female Dermacentor reticulatus and Rhipicephalus appendiculatus ticks induced constriction in a rat femoral artery model; males induced vasoconstriction or vasodilation depending on the time of feeding. Based on comparative HPLC fractionation, the active compounds inducing vasoconstriction do not appear to be prostaglandins (which ticks normally use as vasodilators). Vasoconstriction may be unique to ixodid ticks, helping them control blood flow during their prolonged blood-feeding of up to 10days or more.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues ofTheileria parva antigen genes.

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. ‘Deep 454 pyrosequencing’ of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.

Resistance status of ticks (Acari; Ixodidae) to amitraz and cypermethrin acaricides in Isoka District, Zambia.

This study was designed to obtain data on the farmer's approach to tick control and to determine whether Rhipicephalus appendiculatus Neuman, Amblyomma variegatum (Fabricius), and Rhipicephalus (Boophilus) microplus (Canestrini) were resistant to amitraz and cypermethrin acaricides, in Isoka District, Zambia. Prevailing tick control practices were documented by administering a semi-structured questionnaire to 80 randomly selected smallholder livestock farmers from four agricultural camps (Longwe, Kantenshya, Kapililonga, and Ndeke) in Isoka District. Modified larval packet test (LPT) bioassay experiments were used to determine the resistance status of the common tick species against amitraz and cypermethrin acaricides. Fifty percent of respondents practiced chemical tick control with amitraz (27%) and cypermethrin (23%) being the acaricides in use, and were applied with knapsack sprayers. Less than 3l of spray wash per animal was used which was considerably lower than the recommended delivery rate of 10l of spray wash per animal. No significant susceptibility change to amitraz at 95% confidence level was observed in R. appendiculatus and A. variegatum against amitraz. However, a significant change in the susceptibility of R. (Bo.) microplus tested with amitraz was detected at 95% confidence. The test population had a lower susceptibility (LD50 0.014%; LD90 0.023%) than the reference population (LD50 0.013%; LD90 0.020%). The results indicated that resistance to amitraz was developing in R. (Bo.) microplus. For cypermethrin, no significant susceptibility change at 95% confidence was observed in any of the three species and thus resistance to this chemical was not observed.

Multi-locus genotyping reveals absence of genetic structure in field populations of the brown ear tick (Rhipicephalus appendiculatus) in Kenya

Rhipicephalus appendiculatus is an important tick vector of several pathogens and parasitizes domestic and wild animals across eastern and southern Africa. However, its inherent genetic variation and population structure is poorly understood. To investigate whether mammalian host species, geographic separation and resulting reproductive isolation, or a combination of these, define the genetic structure of R. appendiculatus, we analyzed multi-locus genotype data from 392 individuals from 10 geographic locations in Kenya generated in an earlier study. These ticks were associated with three types of mammalian host situations; (1) cattle grazing systems, (2) cattle and wildlife co-grazing systems (3) wildlife grazing systems without livestock. We also analyzed data from 460 individuals from 10 populations maintained as closed laboratory stocks and 117 individuals from five other species in the genus Rhipicephalus. The pattern of genotypes observed indicated low levels of genetic differentiation between the ten field populations (FST=0.014±0.002) and a lack of genetic divergence corresponding to the degree of separation of the geographic sampling locations. There was also no clear association of particular tick genotypes with specific host species. This is consistent with tick dispersal over large geographic ranges and lack of host specificity. In contrast, the 10 laboratory populations (FST=0.248±0.015) and the five other species of Rhipicephalus (FST=0.368±0.032) were strongly differentiated into distinct genetic groups. Some laboratory bred populations diverged markedly from their field counterparts in spite of originally being sampled from the same geographic locations. Our results demonstrate a lack of defined population genetic differentiation in field populations of the generalist R. appendiculatus in Kenya, which may be a result of the frequent anthropogenic movement of livestock and mobility of its several wildlife hosts between different locations.

Propagation of the Israeli vaccine strain of Anaplasma centrale in tick cell lines

Anaplasma centrale has been used in cattle as a live blood vaccine against the more pathogenic Anaplasma marginale for over 100 years. While A. marginale can be propagated in vitro in tick cell lines, facilitating studies on antigen production, immunisation and vector-pathogen interaction, to date there has been no in vitro culture system for A. centrale. In the present study, 25 cell lines derived from 13 ixodid tick species were inoculated with the Israeli vaccine strain of A. centrale and monitored for at least 12 weeks by microscopic examination of Giemsa-stained cytocentrifuge smears. Infection of 19 tick cell lines was subsequently attempted by transfer of cell-free supernate from vaccine-inoculated tick cells. In two separate experiments, rickettsial inclusions were detected in cultures of the Rhipicephalus appendiculatus cell line RAE25 28–32 days following inoculation with the vaccine. Presence of A. centrale in the RAE25 cells was confirmed by PCR assays targeting the 16S rRNA, groEL and msp4 genes; sequenced PCR products were 100% identical to published sequences of the respective genes in the Israeli vaccine strain of A. centrale. A. centrale was taken through three subcultures in RAE25 cells over a 30 week period. In a single experiment, the Dermacentor variabilis cell line DVE1 was also detectably infected with A. centrale 11 weeks after inoculation with the vaccine. Availability of an in vitro culture system for A. centrale in tick cells opens up the possibility of generating a safer and more ethical vaccine for bovine anaplasmosis.

Distribution of endemic and introduced tick species in Free State Province, South Africa.

The distributions of endemic tick vector species as well as the presence of species not endemic to Free State Province, South Africa, were determined during surveys or opportunistic collections from livestock, wildlife and vegetation. Amongst endemic ticks, the presence of Rhipicephalus appendiculatus was confirmed in the north of the province, whilst Rhipicephalus decoloratus was collected at 31 localities mostly in the centre and east, and Ixodes rubicundus at 11 localities in the south, south-west and centre of the province. Amongst the non-endemic species adult Amblyomma hebraeum were collected from white rhinoceroses (Ceratotherium simum) on four privately owned farms, whilst the adults of Rhipicephalus microplus were collected from cattle and a larva from vegetation at four localities in the east of the province. The collection of Rhipicephalus evertsi mimeticus from a sheep in the west of the province is the second record of its presence in the Free State, whereas the presence of Haemaphysalis silacea on helmeted guineafowl (Numida meleagris) and vegetation in the centre of the province represents a first record for this species in the Free State. The first collection of the argasid tick, Ornithodoros savignyi, in the Free State was made from a domestic cow and from soil in the west of the province. The localities at which the ticks were collected have been plotted and the ticks’ role in the transmission or cause of disease in domestic livestock and wildlife is discussed.

Orcokinin-like immunoreactivity in central neurons innervating the salivary glands and hindgut of ixodid ticks.

Orcokinins are conserved neuropeptides within the Arthropoda but their cellular distribution and functions in ticks are unknown. We use an antibody against the highly conserved N-terminal (NFDEIDR) of mature orcokinin peptides to examine their distribution in six ixodid species: Amblyomma variegatum, Dermacentor reticulatus, Hyalomma anatolicum, Ixodes scapularis, Ixodes ricinus and Rhipicephalus appendiculatus. Numerous immunoreactive neurons (~100) were detected in various regions of the synganglion (central nervous system) in all examined tick species. Immunoreactive projections of two prominent groups of efferent neurons in the post-oesophageal region were examined in detail: (1) neurons innervating the salivary glands; (2) neurons innervating the hindgut. Using matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF), we detected orcokinin peaks in extracts of the synganglia and hindguts but not in the salivary glands of I. scapularis females. Our data provide further evidence of the presence of orcokinin in ixodid ticks and establish a morphological basis for functional studies of identified peptidergic neuronal networks.

First phylogenetic analysis of a Crimean-Congo hemorrhagic fever virus genome in naturally infected Rhipicephalus appendiculatus ticks (Acari: Ixodidae).

Crimean-Congo haemorrhagic fever (CCHF) is a potentially fatal systemic viral disease in many parts of the world, including Iran. The nationwide incidence of human CCHF in endemic areas was 870 confirmed cases with 126 deaths (case fatality rate, CFR = 17.6 %) in the decade leading to 2012. The detection of the CCHF virus (CCHFV) genome in tick vectors is of fundamental importance for identifying these ticks as potential reservoirs of CCHFV infection. From May to October 2013, following detection of four new clinical cases resulting in two deaths in the city of Mashhad (northeast Iran), hard ticks were recovered from infested livestock in 40 villages in Khorasan-Razavi province and examined by the microscopic method for species identification. About a quarter of the ticks were then subjected to reverse-transcription polymerase chain reaction (RT-PCR) to detect the CCHFV genome. The PCR products were then sequenced, and their phylogenetic lineages were determined. A total of 407 hard ticks were captured, representing seven different species in two distinct genera. Members of the genus Hyalomma were widely distributed in all but two of the villages studied, and this was also the most frequent (83.3 %) tick genus. Of 105 adult ticks subjected to RT-PCR, four (3.8 %) ticks were found positive for the CCHFV genome. One brown ear tick, Rhipicephalus appendiculatus, was found to be naturally infected for the first time anywhere in the world. Ticks of Hyalomma asiaticum, Hyalomma marginatum, and Rhipicephalus turanicus were also found to be naturally infected with CCHFV. CCHFV found in these four different tick species were clustered in the same lineage with the Matin and SR3 strains from Pakistan and some other strains from Iran, indicating that these tick species were naturally infected with genetically closely related CCHFV in the region. The presence of CCHFV infection in four different hard tick species was confirmed using RT-PCR in northeast Iran. Part of this infection was attributed to Rh. appendiculatus, which is thus a potential new natural vector of CCHFV in Iran. It is also confirmed by phylogenetic analysis that CCHFV in this region is genetically closely related, even in the different tick species.

An update on the ecological distribution of the Ixodidae ticks in Zimbabwe.

In total 7657 ticks were collected from 121 dip tanks in 12 districts representative of Zimbabwe's five ecological regions between September 2013 and May 2014. Based on morphological traits four genera and 13 species of ticks were identified. Amblyomma hebraeum (60.3%), Rhipicephalus microplus (58.7%), Rhipicephalus decoloratus (47.1%), Rhipicephalus appendiculatus (56.2%), Rhipicephalus evertsi evertsi (67.8%), Rhipicephalus (near) punctatus (13.2%), Hyalomma truncatum (38%) and Hyalomma rufipes (46.3%) were found in all the ecological regions of the country. Amblyomma variegatum and Rhipicephalus compositus (0.8%) were only found in the north central part of the country while Rhipicephalus simus (5%) had a sparse distribution. The Haemaphysalis leachi group (1.7%) and Rhipicephalus sanguineus (1.7%) were found whenever dogs were sampled suggesting these could be widespread throughout the country. The study confirmed the continued limited distribution of A. variegatum (3.3%) in the north central parts of the country, whereas A. hebraeum was found to have a wide distribution also encroaching areas of high rainfall and lower temperatures where it was not previously recorded. A parapatric relationship existed between these two Amblyomma species. Rhipicephalus appendiculatus was also widely distributed although its presence was dominant in the cooler and wetter parts of the country. The traditionally held view that Hyalomma species and R. evertsi evertsi can survive well under diverse conditions is upheld in this study. Rhipicephalus microplus was also present in dry regions but its adaptability to these regions requires further investigation.

Chemical composition and larvicidal activity of essential oil of &lt;i&gt;Lippia kituiensis&lt;/i&gt; against larvae of &lt;i&gt;Rhipicephalus appendiculatus&lt;/i&gt;

This study evaluated the larvicidal activity of essential oil of Lippia kituiensis leaves against larvae of Rhipicephalus appendiculatus. The oil was obtained by hydro-distillation of fresh leaves and analysed using Gas Chromatography-Mass Spectrometry (GC-MS). Analysis showed that sesquiterpenes were dominant (56.57%), followed by monoterpenes (36.36%), diterpenes (2.59%) and others (5.19%). Major sesquiterpenes were germacrene D, β-bourbonene, gamma cadinene and 2-isopropyl-5-methyl-9-methylene-bicycle (4.4.0)dec-1-ene. Major monoterpenes were (1S, 4S)-(-)-camphor, trans-sabinene hydrate, gamma-terpinene, dl-limonene, alpha-terpinolene, l-Phellandrene, beta-Myrcene, sabinene, camphene, alpha-pinene, 4-terpineol, 4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol, 14.29 borneol (=endo-borneol), camphore, and neo-alloocimene. At 6, 12, 24, and 48 hrs after larval treatment, the oil showed activity against R. appendiculatus larvae with LC50in mg/ml of 3.26(3.14-3.38), 3.21(3.08-3.32), 3.15(3.03-3.26), 3.09(2.97-3.20) while LC90in mg/ml were 4.15 (3.95-4.45), 4.03 (3.85-4.30), 3.94 (3.77-4.19), 3.86 (3.69-4.09) respectively. Results of one way ANOVA (Analysis of Variance) showed there was no significant difference in activity of the oil against the larvae, between 6, 12, 24 and 48 hrs in all the concentrations used P = 0.97, 95% confidence. The findings indicatedthat essential oil of L. kituiensis possessed larvicidal properties and can be used to control tick larvae.Keywords: Rhipicephalus appendiculatus; essential oils; acaricidal activity, ticks.

Acaricidal Coumarins from the Medicinal Plant Acokanthera schimperi

Two coumarins were isolated from the plant Acokanthera schimperi (Apocynaceae), and the in vitro larvicidal activities of these compounds against East Coast Fever vector Rhipicephalus appendiculatus was investigated. The mortalities of the larvae were determined after 48 h post treatment. The finding demonstrated that Compound 1 and 2 as amixture registered LC50/LC90 values of 2.96/6.09 mg/ ml. These findings could be useful as lead compounds for the development of acaricides. Additionally, these findings underscore the fact that this plant is used ethnomedicinally as an acaricide. The discovery of plant compounds that may control ticks could be of great value to the agricultural sector.

Efficacy of spot-spray application of Metarhizium anisopliae formulated in emulsifiable extract of Calpurnia aurea in attracting and infecting adult Rhipicephalus appendiculatus ticks in semi-field experiments

The efficacy of spot-spray application of conidia of Metarhizium anisopliae (Metsch.) Sorok. conidia formulated in an emulsifiable extract of Calpurnia aurea (Ait.) Benth in attracting and infecting Rhipicephalus appendiculatus Neumann, 1901 ticks was evaluated in semi-field experiments. Formulation was applied on a 900-cm² spot and ticks released from various distances. Attracted ticks were individually placed in glass vials to determine the number of M. anisopliae conidia picked by a single tick. Infected ticks were either transferred in glass vials or maintained in the laboratory until death or exposed to rabbits in order to investigate the effects of fungal infection on feeding and potential reproduction parameters. Data showed that individual ticks picked between 3.2 and 4.1 × 105 conidia. Mortality of 83 % was observed among ticks maintained in the laboratory conditions. The engorgement period of fungus-infected ticks significantly increased by 16 % compared to the control. A significant reduction (P < 0.0001) was recorded in body weight, egg-mass and egg hatchability from fungus-infected females. It is therefore possible to use M. anisopliae formulated in C. aurea extract for an integrated pest management approach. This study showed that conidia of M. anisopliae formulated in C. aurea extract are effective in attracting, infecting and causing mortalities of R. appendiculatus ticks. Moreover, the reproduction performance of fungus-infected females was significantly reduced.

Antihistamine response: a dynamically refined function at the host-tick interface.

BackgroundTicks counteract host inflammatory responses by secreting proteins from their saliva that compete for histamine binding. Among these tick salivary proteins are lipocalins, antiparallel beta-barrel proteins that sequester small molecules. A tick salivary lipocalin has been structurally resolved and experimentally shown to efficiently compete for histamine with its native receptor (e.g., H1 histamine receptor). To date, molecular dynamics simulations focus on protein-protein and protein-ligand interactions, but there are currently no studies for simultaneous ligand exploration between two competing proteins.MethodsAided by state-of-the-art, high-throughput computational methods, the current study simulated and analyzed the dynamics of competitive histamine binding at the tick-host interface using the available crystal structures of both the tick salivary lipocalin histamine-binding protein from Rhipicephalus appendiculatus and the human histamine receptor 1.ResultsThe attraction towards the tick salivary lipocalin seems to depend on the protonated (adding a hydrogen ion) state of histamine since the current study shows that as histamine becomes more protonated it increases its exploration for the tick salivary lipocalin. This implies that during tick feeding, histamine may need to be protonated for the tick salivary lipocalin to efficiently sequester it in order to counteract inflammation. Additionally, the beta-hairpin loops (at both ends of the tick salivary lipocalin barrel) were reported to have a functional role in sequestering histamine and the results in the current study concur and provide evidence for this hypothesis. These beta-hairpin loops of the tick salivary lipocalin possess more acidic residues than a structurally similar but functionally unrelated lipocalin from the butterfly, Pieris brassicae; comparative results indicate these acidic residues may be responsible for the ability of the tick lipocalin to out-compete the native (H1) receptor for histamine.ConclusionsThree explanatory types of data can be obtained from the current study: (i) the dynamics of multiple binding sites, (ii) competition between two proteins for a ligand, and (iii) the intrinsic molecular components involved in the competition. These data can provide further insight at the atomic level of the host-tick interface that cannot be experimentally determined. Additionally, the methods used in this study can be applied in rationally designing drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0491-9) contains supplementary material, which is available to authorized users.

First molecular detection of Rickettsia africae in ticks from the Union of the Comoros

BackgroundRickettsia africae is the agent of African tick bite fever, a disease transmitted by ticks in sub-Saharan Africa. In Union of the Comoros, a recent study reported the presence of a Rickettsia africae vector but no information has been provided on the circulation of the pathogenic agent in this country.MethodsTo evaluate the possible circulation of Rickettsia spp. in Comorian cattle, genomic DNA was extracted from 512 ticks collected either in the Union of the Comoros or from animals imported from Tanzania and subsequently tested for Rickettsia infection by quantitative PCR.ResultsRickettsia africae was detected in 90% (60/67) of Amblyomma variegatum, 1% (1/92) of Rhipicephalus appendiculatus and 2.7% (8/296) of Rhipicephalus (Boophilus) microplus ticks collected in the Union of the Comoros, as well as in 77.14% (27/35) of Amblyomma variegatum ticks collected from imported cattle. Partial sequences of both bacterial gltA and ompA genes were used in a phylogenetic analysis revealing the presence of several haplotypes, all included within the Rickettsia africae clade.ConclusionsOur study reports the first evidence of Rickettsia africae in ticks collected from the Union of the Comoros. The data show a significant difference of infection rate of Rickettsia africae infected ticks between the Islands, with maximum rates measured in Grande Comore Island, sheltering the main entry port for live animal importation from Tanzania. The high infection levels reported herein indicate the need for an in-depth assessment of the burden of rickettsioses in the Union of the Comoros, especially among those at risk of infection, such as cattle herders.

Identification and sequence characterization of novel Theileria genotypes from the waterbuck (Kobus defassa) in a Theileria parva-endemic area in Kenya

Waterbuck (Kobus defassa), an ungulate species endemic to the Eastern African savannah, is suspected of being a wildlife reservoir for tick-transmitted parasites infective to livestock. Waterbuck is infested by large numbers of Rhipicephalus appendiculatus, the tick vector for Theileria parva, and previous data suggests that the species may be a source of T. parva transmission to cattle.In the present study, a total of 86 cattle and 26 waterbuck blood samples were obtained from Marula, a site in Kenya endemic for East Coast fever (ECF) where the primary wildlife reservoir of T. parva the Cape buffalo (Syncerus caffer) is also common. To investigate for the presence of cattle-infective Theileria parasites, DNA specimens extracted from the blood samples were subjected to two diagnostic assays; a nested PCR based on the p104 gene that is specific for T. parva, and a reverse line blot (RLB) incorporating 13 oligonucleotide probes including all of the Theileria spp. so far described from livestock and wildlife in Kenya. Neither assay provided evidence of T. parva or Theileria sp. (buffalo) infection in the waterbuck DNA samples. By contrast, majority of the cattle samples (67.4%) were positive for T. parva using a nested PCR assay.The RLB assay, including a generic probe for the genus Theileria, indicated that 25/26 (96%) of the waterbuck samples were positive for Theileria, while none of the 11 Theileria species-specific probes hybridized with the waterbuck-derived PCR products. Phylogenetic analysis of 18S ribosomal RNA (18S rRNA) and internal transcribed spacer (ITS) sequences within the RLB-positive waterbuck samples revealed the occurrence of three Theileria genotypes of unknown identity designated A, B and C. Group A clustered with Theileria equi, a pathogenic Theileria species and a causative agent of equine piroplasmosis in domestic equids. However, DNA from this group failed to hybridize with the T. equi oligonucleotide present on the RLB filter probe, suggesting the occurrence of novel taxa in these animals. This was confirmed by DNA sequencing that revealed heterogeneity between the waterbuck isolates and previously reported T. equi genotypes. Group B parasites clustered closely with Theileria luwenshuni, a highly pathogenic parasite of sheep and goats reported from China. Group C was closely related to Theileria ovis, an apparently benign parasite of sheep. Together, these findings provided no evidence that waterbuck plays a role in the transmission of T. parva. However, novel Theileria genotypes detected in this bovid species may be of veterinary importance.