Factors influencing the distribution of questing ticks and the prevalence estimation of T. parva infection in brown ear ticks in the Tanga region, Tanzania.

Questing ticks from various districts and agro-ecological zones (AEZ) in the Tanga Region of Tanzania were studied for a two-year period between September 1999 and July 2001. Collections of both nymphal and adult ticks occurred at 29 sites using a blanket or white cloth dragging technique. The species recorded in the order of decreasing abundance were Rhipicephalus appendiculatus, Boophilus spp., and Ambylomma variegatum. Rhipicephalus appendiculatus field infestation levels varied across administrative districts and seasons, with Amani and the cool-to-dry season having lower tick counts ( = -2.09, SE = 0.71, P < 0.001 and = -1.54, SE = 0.56, P = 0.006 for Amani and cool to dry season, respectively). Based on the polymerase chain reaction technique, Theileria parva infection prevalence in adult R. appendiculatus was estimated to be 2.6%. Implications of these findings in light of the spatial and temporal distribution of ticks are discussed for the accurate diagnosis of multiple tick-borne diseases, the infected vector tick, the endemic status of T. parva in the region, and the implemention of control strategies.     

Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus ticks

A rapid method is described for preparing and staining salivary glands of Rhipicephalus appendiculatus ticks infected with Theileria parva. The technique, involving the use of a modified methyl green pyronin stained minimizes the risk of losing material and allows examination of stained glands within minutes of preparation. The technique was applied in a series of studies in which ticks were either infected with T. parva under different conditions, or maturation of parasites in adult ticks was stimulated by different means. When nymphal ticks were fed on the ears of cattle the subsequent infection rate of the adult ticks showed no correlation with the parasitaemia of the cattle at the time of nymphal engorgement. There was no difference in infection rates between adult ticks in which parasite maturation had been stimulated either by incubation at 37 degree C or by feeding on rabbits. However, parasite maturation took about 1 day longer in incubated ticks than in rabbit-fed ticks. Female ticks were consistently more highly infected than males, both in terms of the percentage of ticks infected and the mean number of infected acini/tick. Ticks were infected with T. parva by injection of nymphs with parasitaemic bovine blood, but the resultant adult infection was lower than that in ticks which had been infected naturally by feeding on cattle.     

Comparative studies on the infectivity of Theileria parva in ticks fed in vitro and those fed on cattle

Nymphs of the brown ear tick, Rhipicephalus appendiculatus, were fed on heparinised bovine blood infected with Theileria parva parasites in an in vitro feeding system consisting of rabbit skin membranes. The main feeding and development parameters such as the mean attachment rate, feeding duration and engorgement weights of membrane-fed ticks were not significantly different from nymphs fed on cattle. The moulting rate was also comparable although a slight significant difference was observed. Assessment of infection prevalence and abundance with T. parva in adults indicated that the membrane-fed ticks acquired infection to the same level as those fed on cattle. Stabilates prepared from both the membrane- and cattle-fed adult ticks were found to be infective and caused severe reactions in susceptible cattle. When the immunised cattle were challenged with a lethal homologous dose of T. parva (Marikebuni), they were found to be immune.     

Variation in body size in the tick complex Rhipicephalus appendiculatus/Rhipicephalus zambeziensis.

We examined the relationship between body size and the phenology of the tick complex Rhipicephalus appendiculatus/Rhipicephalus zambeziensis. These ticks transmit Theileria parva in cattle. In Africa, the body size of R. appendiculatus increases with latitude while the body size of the morphologically similar Rhipicephalus zambeziensis is constant at two different latitudes. A larger body size is necessary once survival becomes a constraint. The most plausible explanation for the smaller R. appendiculatus in equatorial Africa is the cost to produce a larger egg. The consequences of these findings for the introduction of R. appendiculatus in new environments are discussed. New field observations from southern Zambia indicate that R. appendiculatus body size does not vary seasonally as compared to eastern Zambia. This is an additional indication of the presence of a single diapausing population of larger ticks.     

Assessment of Theileria infections in Rhipicephalus appendiculatus ticks collected from the field

Collections of adult Rhipicephalus appendiculatus ticks were made from bait cattle and vegetation at two field sites in areas of Kenya in which East Coast fever caused by Theileria parva is endemic. These ticks, together with two experimentally infected batches of ticks, were examined for infection with Theileria by four methods. Whole salivary glands were stained with methyl green pyronin or Feulgen's stain. Whole ticks were ground in medium, the suspensions were filtered and centrifuged and the treated material was examined microscopically and tested for infectivity by inoculation into cattle. All field collections and experimental batches of ticks were infected with Theileria and all four methods detected the infections. Approximately 1.5% of the ticks in the field collections were found to be infected with Theileria and the treated material from these ticks transmitted T. parva to cattle. It is considered that it will be feasible to survey field infection rates quantitatively by collecting ticks from bait cattle and vegetation for examination by a combination of salivary gland staining and preparation of tick suspensions for microscopy and infectivity tests.     

Genetic variation in the body weight of ticks of the Rhipicephalus appendiculatus/zambeziensis complex

Sixteen strains of ticks of the Rhipicephalus appendiculatus/zambeziensis complex were created by cross-breeding for one to three generations. The body-weights of adult male ticks of these consanguineous lines were compared. A significant genetic variation was distinguished between the groups. Three classes whose mean weights varied around, respectively, 2, 3 and 4 mg/adult male tick, were found. This variation in weight may be explained by hybridization between R. appendiculatus Neumann and R. zambeziensis Walker, Norval & Corwin. Hybrid ticks were undersized whereas R. appendiculatus and R. zambeziensis groups had significantly higher weights.     

Four serine proteinase inhibitors (serpin) from the brown ear tick,Rhiphicephalus appendiculatus; cDNA cloning and preliminary characterization

While development of an anti-Boophilus microplus vaccine is advanced and practical, work on other economically important ticks such as Rhipicephalus appendiculatus is still in its infancy. Guess PCR primers, designed from a consensus amino acid sequence (NAVYKFG) motif were used with rapid amplification of cDNA ends (RACE) to clone four cDNAs encoding serine proteinase inhibitors (serpin) from the brown ear tick Rhipicephalus appendiculatus. The four genes designated as R. appendiculatus serpin (RAS) -1 to -4 encode polypeptides of 378, 380, 398 and 486 amino acids long, respectively. Sequence comparison of RAS-1 to -4 predicted amino acid sequences to the serpin-like hypothetical protein from Ixodes ricinus (Leboulle et al., 2002) revealed closer structural similarities among tick serpins. Expression analysis by RT-PCR showed that RAS-1 to -4 are expressed in other tick organs in addition to salivary glands and midguts. Except for RAS-3 whose expression level appears to be equivalent in all tick organs, RAS-1, -2 and -4 are predominantly expressed in the salivary glands. We have discussed our findings with reference to development of vaccines against R. appendiculatus.     

A cement protein of the tick Rhipicephalus appendiculatus,located in the secretory e cell granules of the type III salivary gland acini,induces strong antibody responses in cattle

Protein components of the cement cone of ixodid ticks are candidates for inclusion in vaccines against tick infestation, since they are essential for tick attachment and feeding. We describe here the cloning of a cDNA encoding a 36 kDa protein, designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36), present in salivary glands and the cement cone material secreted by Rhipicephalus appendiculatus. The 334-amino-acid sequence of RIM36 has a high content of glycine, serine and proline. The protein contains a predicted N-terminal signal peptide and two classes of glycine-rich amino acid repeats, a GL[G/Y/S/F/L] tripeptide and a GSPLSGF septapeptide. Comparison of genomic and cDNA sequences reveals a 597 bp intron within the 3' end of the RIM36 gene. Immuno-electron microscopy demonstrates that RIM36 is predominantly located in the e cell granules of the type III salivary gland acini. An Escherichia coli recombinant form of the proline-rich C-terminal domain of RIM36 reacts with antisera from Bos indicus cattle, either experimentally infested with R. appendiculatus, or exposed to ticks in the field. The 36 kDa protein is strongly recognised on Western blots of salivary gland lysates and soluble extracts of purified R. appendiculatus cement cones by polyclonal antibodies generated against recombinant RIM36, and by antisera from cattle experimentally infested with ticks. The data indicate that this tick cement component is a target of strong antibody responses in cattle exposed to feeding ticks.     

Vector capacity of Rhipicephalus appendiculatus and Amblyomma variegatum for Thogoto and Dhori viruses

ABSTRACT. Experimental studies were undertaken to ascertain the vector potential of Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius for Thogoto (THO) and Dhori (DHO) viruses, candidate members of the Orthomyxoviridae. In the first set of experiments, ticks were infected orally by feeding on viraemic hamsters. THO virus replicated in R.appendiculatus and A.variegatum, persisted trans-stadially in both tick species, and was transmitted to susceptible hosts during feeding. In contrast, both R. appendiculatus and A. variegatum were refractory to per os infection by DHO virus. In the second set of experiments, engorged R. appendiculatus and A. variegatum nymphs were parenterally inoculated with DHO virus. The virus persisted transstadially in both tick species and was transmitted by bite to susceptible hosts. These results indicate that the midgut acts as a barrier to per os infection of R.appendiculatus and A. variegatum by DHO virus. However, when this barrier is bypassed, i.e. by parenteral inoculation, both R.appendiculatus and A.variegatum can serve as efficient vectors of DHO virus.     

Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum

Earlier work from our laboratory indicated that injection of 20-hydroxyecdysone (20E) into non-vitellogenic female Amblyomma hebraeum ticks stimulates the synthesis of vitellogenin (Vg), but not its uptake into oocytes [Friesen, K., Kaufman, W.R., 2004. Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum. Journal of Insect Physiology 50, 519-529]. In contrast, Thompson et al. [Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., Apperson, C.S., Roe, M.R., 2005. In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say). Journal of Insect Physiology 51, 1105-1116] demonstrated that injection of 20E into virgin female Dermacentor variabilis ticks stimulated both vitellogenesis and Vg uptake into oocytes. In addition to the species difference in the two studies there were substantially different methods for injecting 20E. In our earlier work we injected small partially fed ticks after removing them from the host. Thompson et al. injected the females while they remained attached to the host. So in this study we repeated our earlier experiments on A. hebraeum using on-host injection. We also injected 20E into off-host ticks with or without haemolymph collected from engorged ticks (days 2-10 post-engorgement), or from large partially fed mated ticks in the rapid phase of engorgement, to see whether we might detect a 'vitellogenin uptake factor' (VUF) in haemolymph. Off-host injection of 20E (0.45mug/g body weight (bw)) did not induce ovary development beyond that of vehicle-injected controls. But ticks in this study, receiving 20E plus haemolymph from engorged ticks, showed a significant increase in ovary weight beyond that of 20E alone (1.31+/-0.05% bw; 34 for 20E plus haemolymph and 1.03+/-0.05% bw; 25 for 20E alone). However, in normal engorged A. hebraeum, the ovary exceeds 7% bw at the onset of oviposition. As in our earlier work, in this study 20E stimulated Vg-synthesis (3.9+/-0.5mgVt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76+/-0.14mgVt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9+/-1.0mgVt-equivalents/ml). On-host injection of 20E alone (6mug20E/g bw) did not produce a statistically significant increase in oocyte length over that of vehicle-injected controls, whereas on-host injection of 20E plus engorged haemolymph resulted in significantly larger oocytes (261+/-57mum) compared to vehicle-injected controls (132+/-11mum), compared to 20E alone (131+/-12mum), or haemolymph alone (124+/-24mum). There was a marked stimulation of Vg-synthesis by 31mug20E/g bw (6.0+/-1.5mgVt-equivalents/ml) compared to vehicle-injected controls (1.02+/-33mgVt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12+/-3mugVt/mg ovary) or 20E plus haemolymph (56+/-26mugVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5mugVt/mg ovary). There was also a significant effect of 6mug20E/g bw plus engorged haemolymph on ovary weight (1.74+/-0.29% bw) compared to vehicle-injected ticks (0.95+/-0.10% bw), but not compared to ticks injected with 20E alone (1.25+/-0.19% bw). We conclude that at least some of the differences observed between the two laboratories relate to the species difference, and that there is some evidence that the engorged haemolymph of A. hebraeum contains a VUF.     

An assessment of host resistance to ticks on cross-bred cattle in Burundi

Abstract. Groups of cattle of four different cross-breeds (Ankole × Friesian, Ankole × Brown-Swiss, Ankole × Guernsey, Ankole × Sahiwal) plus a group of pure Ankole cattle were immunized against tick-borne diseases: East Coast Fever, using a Theileria parva trivalent vaccine, and anaplasmosis, babesiosis and, subsequently, heartwater. With the exception of two small subgroups of Ankole and Ankole × Sahiwal which were treated by weekly spraying using Delnav, all the groups were exposed to natural field challenge of ticks to quantify the development of host resistance to ticks. The results indicate resistance to the most abundant species, Rhipicephalus appendiculatus , as follows: Ankole > Ankole × Sahiwal > Ankole × Brown Swiss > Ankole × Friesian > Ankole × Guernsey. In both Amblyomma variegatum and Rhipicephalus evertsi evertsi , the manifestation of different levels of resistance between breeds is inapparent. Correlations between total ticks and standard female ticks were good for R. appendiculatus (r = 0.73) and R. evertsi (r - 0.51) but poor for A. variegatum (r = 0.31). Correlations between species were reasonably consistent (r = 0.43-0.59). There was no significant correlation between tick burdens and daily liveweight gain (DLWG) over the. whole period of the study. Of the cross-bred groups, Ankole × Sahiwal, which had the highest level of resistance, had the highest DLWG. In contrast, the Ankole × Guernsey which developed the lowest level of resistance had only marginally lower DLWG, the difference being only 8 g/day. During the first period of exposure to ticks, however, the untreated Ankole × Sahiwal group had markedly lower DLWG, which may indicate that the development and manifestation of resistance is at the expense of productivity.     

Spatial variation of tick abundance and seroconversion rates of indigenous cattle to Anaplasma marginale, Babesia bigemina and Theileria parva infections in Uganda

Tick abundance and seroconversion rates of 640 indigenous cattle in a mixed crop-livestock system in Uganda were investigated in a 14 months longitudinal study. Up to 100% of the cattle in Buyimini, Kubo, Nanjeho, Ojilai and Sitengo villages (high tick challenge zone) were consistently infested with Rhipicephalus appendiculatus, whereas on average 50% of the cattle in Bunghaji, Hitunga and Magoje villages (low tick challenge zone) were inconsistently infested. Likewise, up to 50% of the cattle in Buyimini, Kubo, Nanjeho, Ojilai and Sitengo villages were consistently infested with R. (Boophilus) decoloratus ticks, while on average 30% of the cattle in Bunghaji, Hitunga and Magoje were inconsistently infested. Seroconversion rates of cattle to Anaplasma marginale infection under low tick challenge were higher than those under high tick challenge, but the reverse was true for Babesia bigemina infection. For Theileria parva infection, seroconversion rates of cattle older than 6 months under low tick challenge were significantly higher than those under high tick challenge (P < 0.05). However, the likelihood of occurrence of theileriosis cases among calves (0-6 m) under high tick challenge was 6 times (Odds ratio = 5.82 [1.30-36.37]) higher than under low tick challenge. The high density of anti-tick plants Lantana camara and Ocimum suave that were widespread in villages with low tick challenge, among other factors, was probably the cause for unfavourable tick survival.     

A novel SINE family occurs frequently in both genomic DNA and transcribed sequences in ixodid ticks of the arthropod sub-phylum Chelicerata

Reassociation kinetics and flow cytometry data indicate that ixodid tick genomes are large, relative to most arthropods, containing>or=10(9) base pairs. The molecular basis for this is unknown. We have identified a novel small interspersed element with features of a tRNA-derived SINE, designated Ruka, in genomic sequences of Rhipicephalus appendiculatus and Boophilus (Rhipicephalus) microplus ticks. The SINE was also identified in expressed sequence tag (EST) databases derived from several tissues in four species of ixodid ticks, namely R. appendiculatus, B. (R.) microplus, Amblyomma variegatum and also the more distantly related Ixodes scapularis. Secondary structure predictions indicated that Ruka could adopt a tRNA structure that was, atypically, most similar to a serine tRNA. By extrapolation the frequency of occurrence in the randomly selected BAC clone sequences is consistent with approximately 65,000 copies of Ruka in the R. appendiculatus genome. Real time PCR analyses on genomic DNA indicate copy numbers for specific Ruka subsets between 5800 and 38,000. Several putative conserved Ruka insertion sites were identified in EST sequences of three ixodid tick species based on the flanking sequences associated with the SINEs, indicating that some Ruka transpositions probably occurred prior to speciation within the metastriate division of the Ixodidae. The data strongly suggest that Class I transposable elements form a significant component of tick genomes and may partially account for the large genome sizes observed.     

Resistance and cross-resistance in guinea-pigs and rabbits to immature stages of ixodid ticks

Infestation of guinea-pigs and rabbits with larvae of any one of five species of ticks, Rhipicephalus appendiculatus Neumann, Rhipicephalus evertsi evertsi Neumann, Amblyomma hebrauem Koch, Amblyomma variegatum Fabricius and Ixodes ricinus L., conferred resistance in the animals when exposed to subsequent infestations with the same tick species. Resistance to infestations by other tick species was not observed.     

Skin test to detect resistance of cattle to Rhipicephalus appendiculatus ticks

The evaluation of a skin test to detect acquired resistance to Rhipicephalus appendiculatus Neumann ticks is described. An extract of salivary glands of partially fed female R. appendiculatus was prepared by dissection, sonication and filtration and used for intra-dermal injection. Tests were carried out on twelve calves of known resistance to R. appendiculatus and on twelve naive calves to establish threshold values for positive reactions. Four rabbits naive to ticks were skin tested repeatedly to assess immunogenicity of the test. Reactions to the test at 1 h and at 24 h after injection were significantly correlated with resistance. The correlation was higher with the reactions at 24 h. Rabbits were immunized by the test but the reactions never exceeded the positive threshold. Further development by field testing is recommended.     

Augmentation of host's naturally acquired immunity by solubilized membrane-bound midgut proteins of the tick Rhipicephalus appendiculatus.

Immune resistance of rabbits to the hard tick Rhipicephalus appendiculatus induced by combined tick infestations and immunization with solubilized midgut membrane proteins were compared with resistance due to 1-3 infestations or immunization alone. Results demonstrate that the level of exposure of rabbits to ticks used in the study does not significantly affect the immune expression resulting from immunization and that the latter augments the resistance due to infestation.     

Genes transcribed in the salivary glands of female Rhipicephalus appendiculatus ticks infected with Theileria parva

We describe the generation of an auto-annotated index of genes that are expressed in the salivary glands of four-day fed female adult Rhipicephalus appendiculatus ticks. A total of 9162 EST sequences were derived from an uninfected tick cDNA library and 9844 ESTs were from a cDNA library from ticks infected with Theileria parva, which develop in type III salivary gland acini. There were no major differences between abundantly expressed ESTs from the two cDNA libraries, although there was evidence for an up-regulation in the expression of some glycine-rich proteins in infected salivary glands. Gene ontology terms were also assigned to sequences in the index and those with potential enzyme function were linked to the Kyoto encyclopedia of genes and genomes database, allowing reconstruction of metabolic pathways. Several genes code for previously characterized tick proteins such as receptors for myokinin or ecdysteroid and an immunosuppressive protein. cDNAs coding for homologs of heme-lipoproteins which are major components of tick hemolymph were identified by searching the database with published N-terminal peptide sequence data derived from biochemically purified Boophilus microplus proteins. The EST data will be a useful resource for construction of microarrays to probe vector biology, vector-host and vector-pathogen interactions and to underpin gene identification via proteomics approaches.     

Population dynamics of ticks on indigenous cattle in a pastoral dry to semi-arid rangeland zone of Uganda

Studies on seasonality and population dynamics of ticks on indigenous cattle and their crosses (calves) were carried out in Buruli Ranching Scheme, Nabiswera, Luwero district of Uganda on three treatment groups of animals: group 1 (twice a week dipping), group 2 (once a month dipping) and group 3 (no tick control). During this study, four major species of ticks of economic importance were recorded in decreasing order of abundance: Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, Amblyomma variegatum and Boophilus decoloratus. Of these ticks, a seasonal pattern of activity was only observed in R. appendiculatus ticks, with peak activities occurring during rainy seasons. Greater numbers of ticks were recorded on cows than calves in the three treatment groups, with the exception of A. variegatum where the reverse occurred. The mean numbers of ticks per animal were highly significantly different (p < 0.01) when group 1 animals were compared with group 2 and 3 animals. However, no significant differences (p > 0.05) were observed in mean tick numbers between group 2 and 3 animals. Highly significant differences (p < 0.01) were observed in mean tick numbers on cows and calves (more than 12 months old) in different calving seasons. The state of lactation only affected tick counts on cows in group 1; significantly more ticks (p < 0.01) were observed in lactating than non-lactating cows. Furthermore, significantly greater (p < 0.05) numbers of ticks (with the exception of B. decoloratus) were recorded during the second year of study (March 1992-May 1993) than the first year (January 1991-February 1992), despite lower rainfall during the former period.     

Immune responses of the argasid tick Ornithodoros moubata moubata induced by infection with the filarial worm Acanthocheilonema viteae

Investigations were undertaken to determine whether the tick Ornithodoros moubata moubata mounted a detectable immune response to primary and secondary infections with Acanthocheilonema viteae. Uninfected control tick survival rate was 70%, but only 45% in the primary infection group. Post-secondary infection survival rate (82%) was comparable to controls, indicating that these selected ticks had some protective advantage. Mean A. viteae infective larvae recovery from ticks with secondary infections was 31.4% lower than expected, suggesting the development of immunity. SDS-PAGE of haemolymph for proteins induced post-primary infection yielded a stronger signal at 45 kDa than controls, which was further elevated post-secondary infection. Proteins at 48, 22 and 16 to 18 kDa were detected in haemolymph from infected ticks but not seen from controls. The direct effect of haemolymph on microfilarial viability was examined using a novel in vitro assay; in these preliminary trials no differences were observed in parasite viability when exposed to haemolymph from infected or uninfected groups of ticks.     

Distribution and phenology of ixodid ticks in southern Zambia

Distribution data for epidemiologically important ticks (Acari: Ixodidae) in the Southern Province of Zambia, one of the main cattle areas of the country, are presented. Boophilus microplus (Canestrini) was not recorded in southern Zambia, whereas Boophilus decoloratus (Koch) is present throughout the area. New distribution patterns for less economically important ixodid ticks are also discussed. Southern Zambia is a transition zone because it is the most northern area in Africa where mixed Rhipicephalus appendiculatus Neumann and Rhipicephalus zambeziensis Walker, Norval & Corwin populations were reported. Although a second generation of adult R. appendiculatus/R. zamnbeziensis was encountered, simulations indicated that this phenomenon is very rare in southern Zambia, mainly because of the colder temperatures during the early dry season and lower rainfall. These simulations were supported by a development trial under experimental conditions. Tick body size measurements showed that southern Zambian ticks are larger than eastern Zambian R. appendiculatus. It is hypothesized that body size is related to diapausing intensity in this species. The epidemiological consequences are that a different approach to control Theileria parva (Theiler) (Piroplasmida: Theileriidae) and other tick-borne diseases is needed in southern Zambia, compared to the one adopted in eastern Zambia.