Brown Ear Tick Research Articles

Linking spatial distribution of Rhipicephalus appendiculatus to climatic variables important for the successful biocontrol by Metarhizium anisopliae in Eastern Africa

Cattle production is constantly threatened by diseases like East Coast fever, also known as theileriosis, caused by the protozoan parasite Theileria parva which is transmitted by ticks such as the brown ear tick, Rhipicephalus appendiculatus. To reduce the extensive use of chemical acaricides, fungal-based microbial control agents such as Metarhizium anisopliae have been tested and show promising results against R. appendiculatus both in field and in semi-field experiments in Africa. However, no known endeavors to link the spatial distribution of R. appendiculatus to climatic variables important for the successful application of M. anisopliae in selected East African countries exists. This work therefore aims to improve the successful application of M. anisopliae against R. appendiculatus by designing a temperature-dependent model for the efficacy of M. anisopliae against three developmental stages (larvae, nymphs, adults) of R. appendiculatus. Afterward a spatial prediction of potential areas where this entomopathogenic fungus might cause a significant epizootic in R. appendiculatus population in three selected countries (Kenya, Tanzania, Uganda) in Eastern Africa were generated. This can help to determine whether the temperature and rainfall at a local or regional scale might give good conditions for application of M. anisopliae and successful microbial control of R. appendiculatus.

Essential oils of Tagetes minuta and Tithonia diversifolia affect host location behaviour and on-host attachment site preference of the brown ear tick, Rhipicephalus appendiculatus in the semi-field studies

Effects of essential oils of Tagetes minuta and Tithonia diversifolia (Asteraceae) on orientation behaviour and on-host attachment site preference of newly hatched adult Rhipicephalus appendiculatus were evaluated. Host animals were treated at the ear pinna (by smearing the oil directly on the ear or suspending a tube containing the oil on the ear pinna) and legs + tail in semi-field plots. The legs + tail sites of the essential oil application showed the lowest mean percentage of ticks observed on host body (16.5 ± 1.9% and 26.0 ± 2.8%) and the highest mean percentage reduction of attaching ticks (76.5 ± 3.9% and 67.0 ± 0.8%) for essential oils of Tagetes minuta and Tithonia diversifolia, respectively. The control animals had the highest mean percentage of ticks observed (93.0 ± 2.1%). The ear tube resulted in the highest mean percentage of ticks on the host (47.5 ± 5.1% and 55.8 ± 5.1%) and a lowest mean percentage reduction of attaching ticks (44.8 ± 5.1% and 36.5 ± 7.4%) for the essential oils of Tagetes minuta and Tithonia diversifolia, respectively. For both the essential oils, legs + tail sites of essential oil application, followed by ear smear and then ear tube, had significant effects on orientation to the host and attachment site preference of adult R. appendiculatus on the host, in that order. We, therefore, recommend the ear smear site for treating hosts with essential oils whose performance may improve upon increasing concentration and formulating them in a carrier material that stabilizes the active ingredients.

Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus

Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail.

Structure-activity studies on analogues of 4-methylguaiacol, a cattle anal odour constituent repellent to the brown ear tick (Rhipicephalus appendiculatus)

Previously, 4-methylguaiacol, a major constituent of cattle anal odour, was found to have a high repellency on Rhipicephalus appendiculatus. In the present study, 10 structural analogues of the phenol were tested for repellency against R. appendiculatus in order to assess the effects of (i) absence or presence of the 4-alkyl group of varying length, (ii) inclusion of a double bond in the 4-alkyl chain, (iii) linking the two phenolic oxygen in a methylenedioxy bridge, (iv) replacement of the OCH3 with CH3 and inclusion of another CH3 at position 6, and (v) presence of an additional OCH3 group at position 6. The analogues comprised of 2-methoxyphenol (guaiacol), 4-ethyl-2-methoxyphenol, 4-propyl-2-methoxyphenol, 4-allyl-2-methoxyphenol (eugenol), 3,4-methylenedioxytoluene, 2,4-dimethylphenol, 4-ethyl-2-methylphenol, 2,4,6-trimethylphenol, 4-propyl-2,6-dimethoxy-phenol and 4-allyl-2,6-dimethoxyphenol, which were compared at different concentrations in a two-choice climbing assay set up. Each analogue showed either increased or reduced repellency compared with 4- methylguaiacol. The structural feature that was associated with the highest repellency was 4-propyl moiety in the guaiacol unit (RD75 = 0.031 for 4-propyl-2-methoxyphenol; that of 4-methylguaiacol = 0.564). Effects of blending selected analogues with high repellency were also compared. However, none of the blends showed incremental increases in repellency compared with that of 4-propyl-2-methoxyphenol. We are currently evaluating the effects of controlled release of this compound at different sites on cattle on the behavior and success of R. appendiculatus to locate their predilection for feeding sites.

Characterisation of cattle anal odour constituents associated with the repellency of Rhipicephalus appendiculatus.

Adults of the Brown Ear Tick (Rhipicephalus appendiculatus) have a predilection for feeding inside and around the ears of cattle and other hosts. A previous study has shown that the tick locates the host ears by 'push-pull' deployment of a repellent blend emitted at the anal region and an attractant blend emitted at the ears. Interestingly, the two odours play reverse roles with Rhipicephalus evertsi, which prefer to feed around the anal region. The present study was undertaken to characterize the major constituents of the cattle anal odour and to evaluate their repellence to R. appendiculatus. The anal odour was trapped with reverse-phase C18-bonded silica, Porapak Q and Super Q placed in an oven bag attached at the anal region of the cattle for 6h. The adsorbents were then removed and extracted with dichloromethane, and the extracted compounds analyzed by linked gas chromatography-mass spectrometry (GC-MS). The major constituents of the odour were o-xylene, 4-hydroxy-4-methyl-2-pentanone, 4-methyl-2-methoxyphenol, ethylbenzene, 2,6,6-trimethyl-[1S(1α,β,5α)]bicycloheptanes, 5-ethoxydihydro-2(3H)-furanone, 3-methylene-2-pentanone, 5-methyl-2-phenyl-1H-indole, and 3-pentanone. The repellency of the available compounds (o-xylene, 4-hydroxy-4-methyl-2-pentanone, 4-methyl-2-methoxyphenol, ethyl benzene, 3-methylene-2-pentanone, and 3-pentanone) and blends was evaluated using a dual choice tick climbing assay at different doses. The anal odour showed repellence with RD75 of 0.39. Of the compounds tested, 4-methyl-2-methoxyphenol was found to be most repellent (RD75 = 0.56) and 3-pentanone least repellent (RD75 = 622.7). The blend of the six constituents showed RD75 of 0.34, comparable to that of the crude anal odour blend. A series of subtractive bioassays with one constituent of the 6-component blend missing was also carried out. Subtraction of 3-methylpentanone gave the most repellent blend (RD75 = 0.097), whereas subtraction of 4-methylguaiacol gave the least repellent blend (RD75 = 160.7) consistent with the high individual activity of this phenol. The study lays down useful groundwork for on-host deployment of controlled-release of a selected repellent or blend to disrupt the tick's ability to locate its preferred feeding site.

Structural basis of cholesterol binding by a novel clade of dendritic cell modulators from ticks

Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.

English

The effects of 2 Neem derivatives, Neem oil (NO) and Neem Seed Powder (NSP), on various physiological parameters of the brown ear tick, Rhipicephalus appendiculatus, the vector for Theileria parva. Neem oil was applied on rabbit ears using a fine brush, whereas NSP was mixed with rabbit pellets at various concentrations and fed on goats on which various instars of the tick were allowed to feed. Tick larvae smeared with Neem oil (NO) while attached to rabbit ears exhibited significant mortalities. The 10% NO induced a mortality of 40% compared to less than 1% observed in Peanut oil (PO) control. Furthermore, engorgement weights were significantly reduced (0.1 mg NO vs 4 mg PO). Their corresponding moulting percentages were 1 and 71% in NO and PO, respectively. Adult female ticks exhibited reduced engorgement weights and egg mass. Larvae able to attach on goats maintained on 100% NSP were only 11% compared to 66% for control goats fed on rabbit pellets (RP). The corresponding nymphal attachments were 24% NSP and 79% RP. The number and weights of eggs produced by adult ticks fed on goats maintained on NSP also dropped significantly. The hatchability of eggs produced by these ticks was also significantly reduced. Key words: East Coast Fever, economic losses, Neem derivatives, Rhipicephalus appendiculatus, Theileria parva, tick control.

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

Paralog analyses reveal gene duplication events and genes under positive selection in Ixodes scapularis and other ixodid ticks.

BackgroundHard ticks (family Ixodidae) are obligatory hematophagous ectoparasites of worldwide medical and veterinary importance. The haploid genomes of multiple species of ixodid ticks exceed 1 Gbp, prompting questions regarding gene, segmental and whole genome duplication in this phyletic group. The availability of the genome assembly for the black legged tick, Ixodes scapularis, and transcriptome datasets for multiple species of ticks offers an opportunity to assess the contribution of gene duplication to the genome. Here we developed a bioinformatics pipeline to identify and analyze duplicated genes (paralogs) using gene models from the prostriate tick, I. scapularis IscaW1.1 annotation and expressed sequence tags (ESTs) from I. scapularis and the metastriate ticks, Rhipicephalus microplus (southern cattle tick), R. appendiculatus (brown ear tick) and Amblyomma variegatum (tropical bont tick).ResultsApproximately 1-2 % of I. scapularis gene models and 2-14 % of ESTs from the four species represent duplicated genes. The ratio of non-synonymous to synonymous nucleotide substitution rates suggests ~ 25 % of duplicated genes are under positive selection pressure in each species. Analyses of synonymous substitution rates provide evidence for two duplication events in I. scapularis and R. microplus involving several hundred genes. Conservative molecular clock estimates based on synonymous substitution rates for species of Anopheles mosquitoes and the fruit fly, Drosophila melanogaster, suggest these events occurred within the last 50 MYA. Mapping of paralogs to the I. scapularis genome assembly supports tandem, or possibly segmental duplication events.ConclusionsThe present study marks the first genome-level analyses of gene duplication for the Ixodidae and provides insights into mechanisms shaping genome evolution in this group. At least two duplication events involving hundreds of genes may have occurred independently in the lineages prostriata and metastriata, with tandem and segmental duplication the most likely mechanisms for paralog generation. Duplicated genes under positive selection pressure may be linked to emerging functions in the tick and represent important candidates for further study.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2350-2) contains supplementary material, which is available to authorized users.

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.

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.

Repellent Activities of Essential Oils of Some Plants Used Traditionally to Control the Brown Ear Tick,Rhipicephalus appendiculatus

Essential oils of eight plants, selected after an ethnobotanical survey conducted in Bukusu community in Bungoma County, western Kenya (Tagetes minuta, Tithonia diversifolia, Juniperus procera, Solanecio mannii, Senna didymobotrya, Lantana camara, Securidaca longepedunculata, and Hoslundia opposita), were initially screened (at two doses) for their repellence against brown ear tick, Rhipicephalus appendiculatus, using a dual-choice climbing assay. The oils of T. minuta and T. diversifolia were then selected for more detailed study. Dose-response evaluations of these oils showed that T. minuta oil was more repellent (RD50 = 0.0021 mg) than that of T. diversifolia (RD50 = 0.263 mg). Gas chromatography-linked mass spectrometric (GC-MS) analyses showed different compositions of the two oils. T. minuta oil is comprised mainly of cis-ocimene (43.78%), dihydrotagetone (16.71%), piperitenone (10.15%), trans-tagetone (8.67%), 3,9-epoxy-p-mentha-1,8(10)diene (6.47%), β-ocimene (3.25%), and cis-tagetone (1.95%), whereas T. diversifolia oil is comprised mainly of α-pinene (63.64%), β-pinene (15.00%), isocaryophyllene (7.62%), nerolidol (3.70%), 1-tridecanol (1.75%), limonene (1.52%), and sabinene (1.00%). The results provide scientific rationale for traditional use of raw products of these plants in controlling livestock ticks by the Bukusu community and lay down some groundwork for exploiting partially refined products such as essential oils of these plants in protecting cattle against infestations with R. appendiculatus.

High-resolution predictive mapping for Rhipicephalus appendiculatus (Acari: Ixodidae) in the Horn of Africa

The brown ear tick Rhipicephalus appendiculatus, vector of East Coast fever (ECF) and related cattle diseases caused by Theileria parva has never been reported from the Horn of Africa. Habitat suitability for this tick species was predicted using Maxent modelling technique based on R. appendiculatus records in Sub-Saharan Africa. Two models were developed: the first is based on the tropical R. appendiculatus distribution and the one is based on the distribution records in the temperate region of Sub-Saharan Africa. The tropical model shows favourable habitat in much of the Ethiopian highlands. The whole Djibouti, the south eastern Ethiopian lowlands, majority of Somalia and Eritrea were found to be not suitable for the survival and development of this tick species. Highly suitable areas occur in areas which have moderate temperature and high precipitation. Introductions of R. appendiculatus into the Horn of Africa probably have been prevented by the natural barrier between the known R. appendiculatus distribution range in East Africa and the Horn of Africa. The effect of an introduction of R. appendiculatus and thereby ECF into the Horn of Africa could be catastrophic since the cattle in this area have no immunity against ECF, and mortality might be considerable in all age groups of cattle.

Acaricidal activities of extracts of Commiphora swynnertonii Burtt (Burseraceae), Melia volkensii Gürke, Turraea abyssinica Hochst., Turraea floribunda Hochst and Turraea cornucopia Styles & F. White (Meliaceae) against the brown ear tick Rhipicephalus ap

Acaricidal activities of extracts of Commiphora swynnertonii Burtt (Burseraceae), Melia volkensii Gurke, Turraea abyssinica Hochst., Turraea floribunda Hochst and Turraea cornucopia Styles & F. White (Meliaceae) against the brown ear tick Rhipicephalus appendiculatus Neumann Extracts of the stem bark of Commiphora swynnertonii, the root bark of Turraea abyssinica, T. cornucopia, T. floribunda and the ripened fruits of Melia volkensii were tested for acaricidal activities against nymphal and adult stages of Rhipicephalus appendiculatus in the laboratory. At a concentration of 10% (w/v), hexane and ethyl acetate extracts of C. swynertonii induced mean mortalities of 71% and 54% respectively, in two week old nymphs. A 10% (w/v) hexane extract was found to be as effective as the commercial acaricide Triatix against the twoweek- old nymphs. Extracts of T . cornucopia, T. abyssinica and M. volkensii induced no mortality in nymphs and adults, but suppressed oviposition capacity during the first day. On the other hand, 500µl of 10% (w/v) methanol extracts of T. cornucopia and T. floribunda delayed attachment by adult females of R. appendiculatus on the rabbit ears, engorgement and hatchability of their eggs. Keywords : acaricidal, activity, plant extracts, Burseraceae, Meliaceae, Rhipicephalus appendicullatus.

Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4a S,7 S,7a R) and (4a S,7 S,7a S)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC–mass spectrometry (GC–MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4a S,7 S,7a R) and (4a S,7 S,7a S)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4a S,7 S,7a R) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4a S,7 S,7a R) and (4a S,7 S,7a S) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon ( E)-(1 R,9 S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD 50 = 0.081 mg cm −2 and chemotype B RD 50 = 0.091 mg cm −2) for An. gambiae comparable with the synthetic repellent DEET (RD 50 = 0.12 mg cm −2), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD 50 = 0.34 mg cm −2 and chemotype B RD 50 = 0.074 mg cm −2). Further repellency testing against An. gambiae using the purified (4a S,7 S,7a R) and (4a S,7 S,7a S)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4a S,7 S,7a R) and (4a S,7 S,7a S) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4a S,7 S,7a R) and (4a S,7 S,7a S) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including ( E)-(1 R,9 S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD 50 = 0.005 mg and chemotype B RD 50 = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as ( E)-(1 R,9 S)-caryophyllene.

Comparison of Entropic Contributions to Binding in a “Hydrophilic” versus “Hydrophobic” Ligand−Protein Interaction

In the present study we characterize the thermodynamics of binding of histamine to recombinant histamine-binding protein (rRaHBP2), a member of the lipocalin family isolated from the brown-ear tick Rhipicephalus appendiculatus. The binding pocket of this protein contains a number of charged residues, consistent with histamine binding, and is thus a typical example of a “hydrophilic” binder. In contrast, a second member of the lipocalin family, the recombinant major urinary protein (rMUP), binds small hydrophobic ligands, with a similar overall entropy of binding in comparison with rRaHBP2. Having extensively studied ligand binding thermodynamics for rMUP previously, the data we obtained in the present study for HBP enables a comparison of the driving forces for binding between these classically distinct binding processes in terms of entropic contributions from ligand, protein, and solvent. In the case of rRaHBP2, we find favorable entropic contributions to binding from desolvation of the ligand; however, the overall entropy of binding is unfavorable due to a dominant unfavorable contribution arising from the loss of ligand degrees of freedom, together with the sequestration of solvent water molecules into the binding pocket in the complex. This contrasts with binding in rMUP where desolvation of the protein binding pocket makes a minor contribution to the overall entropy of binding given that the pocket is substantially desolvated prior to binding.

Tick communities at the expanding wildlife / cattle interface in the Eastern Cape Province, South Africa : implications for Corridor disease

Corridor disease, transmitted by the brown ear tick (Rhipicephalus appendiculatus), is one of Africa's most pathogenic tick-borne diseases for cattle. With a focus on this species, we investigated the community parameters (richness, diversity and abundance) of ticks in the Eastern Cape, South Africa, and how this may be linked to the increasing wildlife/cattle interface in the region. There were significantly more ticks of a greater diversity and richness at sites positioned at the wildlife/cattle interface ('treatment sites') compared to sites where wildlife was absent (controls). Significantly, R. appendiculatus was only found at the treatment sites. Therefore, it is believed that the wildlife/cattle interface may be playing a crucial role in increasing the occurrence, abundance and distribution of R. appendiculatus in the Eastern Cape. The implications of a Corridor disease outbreak in the region are discussed.

Functional role of 64P, the candidate transmission-blocking vaccine antigen from the tick, Rhipicephalus appendiculatus

Anti-ectoparasite vaccines offer attractive alternatives to the use of chemical pesticides, especially if they also control the pathogens that ectoparasites transmit. However, selection of suitable antigens is a major constraint on vaccine development. The recombinant tick cement protein, 64TRP, derived from the African brown ear tick, Rhipicephalus appendiculatus, acts as a transmission-blocking vaccine in a mouse model of tick-borne encephalitis virus (TBEV) transmission, protecting immunised mice against lethal challenge with TBEV after exposure to infected ticks. 64TRP acts as a dual action vaccine, targeting both ‘exposed’ antigens in tick saliva and ‘concealed’ antigenic epitopes in the tick midgut. To assess further the suitability of 64TRP as a vaccine antigen, we examined the function (including localisation) of the protein, and its sequence variability. Histological profiles of normal hamster skin showed similarities between normal skin proteins in the epidermis (keratin) and dermis (collagen/reticulin) and the tick cement cone. Immuno-reactivity of anti-64TRP sera with hamster skin suggests a potential sequence similarity of 64P with host skin proteins and may reflect previously reported sequence similarities of 64P with skin keratin and collagen proteins. Variability in the N-terminal signal peptide and in the C-terminal glycine-rich amino acid repeats of 64P protein was detected; previous studies showed the C-terminal region to be immunologically non-protective. Using in situ hybridisation and quantitative reverse transcriptase-PCR, 64P mRNA was detected in the types II and III salivary gland acini. The highest levels of 64P mRNA were observed in 1-day fed females, and 1- and 7-day fed males. Salivary glands of longer feeding females and unfed ticks as well as midguts of both sexes were negative. Early expression in tick salivary glands is consistent with previously published data that 64P is a cement protein, and contributes to its candidacy as a vaccine antigen. However, further studies are required to assess whether cross-reactivity with skin proteins may induce autoimmunity.

An epidemiological model of East Coast Fever in African livestock

An epidemiological model of the dynamics of East Coast Fever (ECF) in East Africa caused by the protozoan parasite Theileria parva and transmitted by the brown-ear tick Rhipicephalus appendiculatus was developed. In the model, ticks are assigned to either on-host or off-host categories both of which differ in their capacity to receive and transmit the disease. Cattle are assigned to categories of susceptible, infected and infectious as well as recovered animals having immunity to the disease. The parameters of the model were estimated from data reported in the literature. A mathematical analysis of the ECF/tick/cattle model with and without disease was conducted. Depending on the ratio between fecundity and mortality rates in cattle and in the absence of disease, different scenarios emerge including extinction of ticks, coexistence of ticks and cattle and total extinction of ticks and cattle. Furthermore, the analysis of the model with the disease yielded threshold conditions for the existence and the persistence of stable coexistence equilibria for the epidemiological system that may lead to enzootic stability. The model was used to identify critical aspects of the dynamics required to develop management strategies: (i) tick control in areas where the disease is absent, (ii) threshold-based tick and disease control, and (iii) conditions permitting the establishment of enzootic stability of the ECF/tick/cattle system. The analysis also identifies critical areas requiring further field investigation, sets the basis for developing a realistic model for field implementation, and provides a tool for project development and evaluation in the context of international research.

An Ion-channel Modulator from the Saliva of the Brown Ear Tick has a Highly Modified Kunitz/BPTI Structure

Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 Å resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by β-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the ”Kunitz head” by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a β-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.