Specificity and effects of host resistance on the African tickRhipicephalus zambeziensis (Acarina: Ixodidae)

Repeated infestations of the rabbit with adultRhipicephalus zambeziensis ticks resulted in a tenfold reduction in engorgement weight of adult ticks and a significant decline in engorgement weight of nymphs. There was no evidence of cross-resistance against the South African bont tick,Amblyomma hebraeum. Immunization of rabbits with crude extracts of whole-tick protein significantly influenced engorgement weights and oviposition rates of adultR. zambeziensis.Histological studies of tissues fromR. zambeziensis adults fed on resistant rabbits showed severe gut damage; this was associated with the binding of host IgG to mid-gut cells.     

Immunity of the ox to the brown ear tickRhipicephalus appendiculatus

Controlled repeated infestation ofBos taurus andBos indicus cattle resulted in a decline in the recovery of allR. appendiculatus instars. Resistance to infestation varied between individuals although all individuals manifested severe hypersensitivity at the attachment site, which was reflected in the grooming reflex.     

Monitoring of naturally acquired and artificially induced immunity toAmblyomma variegatum andRhipicephalus appendiculatus ticks under field and laboratory conditions

The ability of rabbits, goats and cattle to acquire immunity to the ixodid ticksAmblyomma variegatum andRhipicephalus appendiculatus was studied under laboratory and field conditions. Rabbits were successfully immunized with crude salivary gland extract (SGE) and midgut extract (ME) obtained from flat or partly fed femaleR. appendiculatus ticks. The lowest numbers of larvae were produced by females fed on rabbits immunized with unfed midgut extract. Similar reductions in larval production could be induced after three infestations of rabbits with adultR. appendiculatus. Also, successive feedings of nymphs ofR. appendiculatus on rabbits resulted in significantly reduced engorgement weights. Skin testing with SGE induced delayed-type hypersensitivity reactions, which could be correlated with immunity toR. appendiculatus in rabbits. Moreover, circulating antibodies were detected in rabbits with an ELISA using SGE ofR. appendiculatus.Immunity toA. variegatum nymphs could be induced in rabbits by repeated infestations, but this failed in goats. Immunization of goats with midgut extract from adultA. variegatum did not protect against subsequent nymphal challenge, but strong skin reactions were noticed when adults ticks fed on immunized goats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of SGE and ME fromA. variegatum revealed the presence of 48 protein bands in SGE and 29 bands in midgut extract. Western blotting employing serum from a rabbit immune toR. appendiculatus recognized a number of bands in SGE fromR. appendiculatus, but also in SGE ofA. variegatum.Immunity acquired by cattle to ixodid tick infestations under field conditions was monitored by skin testing with SGE and western blot analysis. In general, cattle with the lowest tick numbers manifested the strongest delayed-type hypersensitivity responses. Finally, western blot analysis employing sera from tick-infested and tick-naive cattle could not be related to actual immune status.     

Biology ofRhipicephalus appendiculatus andR. zambeziensis and production of a fertile hybrid under laboratory conditions

In some areas of Eastern and Southern provinces of ZambiaRhipicephalus appendiculatus andR. zambeziensis are sympatric, and numerous intermediate forms as well as typicalR. appendiculatus andR. zambeziensis are found. Experiments were carried out in order to clarify the taxonomic status of the intermediate forms. Biology and hybridization capacities of two strains ofR. appendiculatus (A and D) and one strain ofR. zambeziensis (H) were studied. Infestations on rabbits and calves were performed at the beginning and at the end of the Zambian rainy season. The adults of both species showed preference for the bovine host and performed better at the end of the rainy season. A recently isolated strain ofR. appendiculatus had notably higher larval production per unit engorgement weight than the strain that had been maintained for prolonged periods under laboratory conditions.R. zambeziensis produces fewer larvae and had longer development periods thanR. appendiculatus.The two species cross-breed under laboratory conditions. A fertile hybrid is produced byR. zambeziensis females andR. appendiculatus males, whereas a sterile hybrid is a result of a reciprocal cross. Morphology of adults of the fertile hybrid (H/A) is indistinguishable from the maternal species, whereas adults of the sterile hybrid (A/H) are of maternal, paternal and intermediate forms.Larval production indices (LPI) vary from 0.4 to 3.7 for the H () x A () cross, approximately three-fold less than in the maternal species. For the reciprocal cross, LPI ranges from 0.1 to 2.7, five- to thirty-fold less than in the maternal species. The fertile hybrid has a higher LPI than both first crosses, and under the most favourable climatic conditions LPI is even higher than for the maternal species.Adults of the sterile hybrid rarely formed pairs. Only partially engorged female ticks detached, which produced no viable eggs. Both hybrids may occur in areas where the two species are sympatric. However, detailed field studies will have to be performed in order to gain knowledge on population dynamics, interbreeding and the vector role of the two species and their hybrids under field conditions.     

Resistance and cross-resistance in rabbits to adults of three species of African ticks (Acari: Ixodidae)

Resistance to Rhipicephalus appendiculatus, Amblyomma variegatum and Amblyomma hebraeum was investigated in the laboratory by infesting rabbits with adults of each of the three species followed by homospecific or heterospecific secondary infestations. Significantly lower female engorged weights and egg mass weights were taken as evidence of protective immunity. Following a single infestation with adults, rabbits developed homospecific protective immunity (resistance) to only R. appendiculatus and A. hebraeum; primary infestation with A variegatum did not protect against secondary infestation with the same species. There was no cross-resistance (heterospecific protective immunity) between the species except for one-way protection between R. appendiculatus and A. variegatum; primary infestation with R. appendiculatus protected against secondary infestation with A. variegatum, but not vice versa. The results from ELISA did not indicate any correlation between serum antibodies to soluble antigens from salivary gland extracts and protective immunity. Post-infestation sera from rabbits infested with each of the three species reacted strongly to their respective salivary gland extracts. Despite the high reactivity of A. variegatum serum with salivary gland antigens from all three species, A. variegatum-infested rabbits did not show any homospecific or heterospecific immunity; on the other hand, although R. appendiculatus serum did not react positively to A. variegatum antigens, infestation with R. appendiculatus protected against a subsequent A. variegatum infestation.     

Immunisation of rabbits against the brown ear tick,Rhipicephalus appendiculatus using tick haemolymph

Tick naive rabbits were immunised with haemolymph components from partially fed Rhipicephalus appendiculatus adult ticks and subsequently challenged with all the developmental instars of the tick. The results obtained showed that the rabbits were rendered resistant to all the instars of the tick. However, the resistance was more pronounced in adult ticks than in the immature stages. The resistance was manifested as a reduction in the number of ticks that fed successfully to engorgement, reduced engorgement weights and reduced fecundities. Re challenging the resistant experimental rabbits with all the developmental instar stages of Rhipicephalus appendiculatus showed that resistance was maintained in subsequent infestations.     

Survival and behaviour of unfed stages of the ticksRhipicephalus appendiculatus,Boophilus decoloratus andB. microplus under field conditions in Zimbabwe

The survival and behaviour of the unfed stages ofRhipicephalus appendiculatus, Boophilus decoloratus andB. microplus in gauze columns were observed in long and short grass in the highveld of Zimbabwe. Ticks were exposed in the cool, hot and rainy seasons of 1980 and 1981. All species and stages survived longer in long grass than in short grass. Larvae from engorged female ticks released in the cool season hatched much later than incubator-reared controls. They were consequently not present during the cold weather and survived longer than larvae subjected to the low temperatures, in which the shortest survival-times were recorded. The survival of nymphs was insensitive to season. The longest survival-times were recorded in adults. Median survival-times of incubator-reared adults ranged from 165 to 375 days in short grass and from 333 to 493 days in long grass. These times were usually longer than those for adults which moulted in the field. Larvae of the three species and nymphs ofR. appendiculatus were active soon after hatching or moulting, irrespective of the season. In contrast, adults ofR. appendiculatus showed different patterns of activity in different seasons. Adults first appeared at the base of the columns in October/November and then gradually ascended to reach a maximum height in December/January. They remained high up in the columns until May/June when the weather became increasingly cold and dry. Larvae ofB. decoloratus climbed higher up in the columns in the long grass than did the larvae of the other two species.Larvae and nymphs ofR. appendiculatus and larvae ofB. microplus migrated up and down the columns daily, but larvae ofB. decoloratus and adults ofR. appendiculatus did not migrate.     

The effect of rainfall on tick challenge at Kyle Recreational Park,Zimbabwe

The effect of rainfall pattern on tick challenge was investigated at Kyle Recreational Park, Zimbabwe, from 1991 to 1992 using drag and removal plot methods to sample environmental tick density. The abundance of adults and nymphs of the brown ear-tick Rhipicephalus appendiculatus and larvae of the bont tick Amblyomma hebraeum was positively correlated with monthly rainfall, whereas no relationship with rainfall was revealed for larval R. appendiculatus, adults of the red-legged tick R. evertsi, or larvae of the blue tick Boophilus decoloratus. A comparison between 1991 (490 mm rainfall) and the drought year of 1992 (161 mm) revealed significant differences in the abundance of R. appendiculatus, A. hebraeum, and B. decoloratus. During the wet season, R. appendiculatus adults were 2–3 time more numerous in the environment during the higher rainfall year of 1991. A. hebraeum larval abundance exhibited a similar pattern to that of R. appendiculatus adults, but B. decoloratus larvae were more abundant in the drought year of 1992 during both the wet and dry seasons. Comparable tick abundance data collected at Kyle during the above-average rainfall years of 1975–1977 (mean = 1029 mm) were compared with tick challenge during the below-average rainfall years of 1991–1992 (mean = 326 mm). In grassland sand habitat and all habitats combined R. appendiculatus adults, nymphs, and larvae were much more abundant during the high rainfall years. In contrast, larvae of B. decoloratus were more numerous during the drier years. A. hebraeum larvae were also more abundant during the drier years. The strong positive correlation of adult R. appendiculatus abundance with rainfall and the coincidence of increased adult tick challenge with increased rainfall indicates that adult R. appendiculatus tick burden on hosts would be heaviest during the wet season and high rainfall years.     

Interstadial and Infestation Level-dependent Variation in the Transmission Efficiency of Borrelia Burgdorferi from Mice to Ixodes Ricinus Ticks

The efficiency with which the spirochaete Borrelia burgdorferi sensu stricto was transmitted from laboratory mice to larval and nymphal Ixodes ricinus ticks was assessed, using the polymerase chain reaction. The transmission efficiency to nymphs was significantly greater than to larvae when both fed together on the same host. Increased tick infestation levels of mice were correlated with significantly greater engorgement weights and higher B. burgdorferi transmission coefficients from mice to nymphs. These observations indicate that both the feeding success of ticks and the transmission coefficients from host to tick may be influenced by the tick infestation level of an infected host. The infestation level and the relative numbers of each life stage of the tick are factors which should be considered in the design of transmission experiments.     

Rhipicephalus appendiculatus and R. zambeziensis (Acari: Ixodidae) from Zambia: a molecular reassessment of their species status and identification

The closely related species Rhipicephalus appendiculatus and R. zambeziensis co-occur in a wide area in Zambia. In this area, specimens of both species have been collected on the same individual host at the same time. In addition, specimens that are morphologically intermediate between R. appendiculatus and R. zambeziensis have been found in this area. These observations cast some doubt on the species status of R. appendiculatus and R. zambeziensis. Because the two taxa have varied influences on the epidemiology of East Coast fever (ECF), a cattle disease for which they act as vectors, it is essential that their taxonomic status is clarified and their identification is accurate. Therefore a phylogenetic analysis of nucleotide sequences of the ITS2 and a fragment of the mitochondrial 12S rDNA was performed to reassess the specific status of both taxa. This revealed two well supported clades coinciding with R. appendiculatus and R. zambeziensis as two separate species. In order to facilitate species identification a PCR-RFLP diagnostic assay was developed based on BauI digestion of the ITS2 gene. This assay produced clear diagnostic banding patterns for the two species and is applicable over a wide range of both species’ distribution. Nucleotide sequence data reported in this paper are available in the GenBank™ under the accession numbers DQ849203–DQ849274.     

Amplification of tick-borne encephalitis virus infection during co-feeding of ticks

Abstract. Following engorgement of Rhipicephalus appendiculatus larvae on guinea-pigs infected with tick-borne encephalitis (TBE) virus, none of the engorged larvae or emergent nymphs contained detectable infectious virus. However, one of twelve pools, each containing three of the unfed nymphs, was positive when screened by polymerase chain reaction (PCR), indicating a low prevalence of TBE virus infection in the unfed nymphs. After engorgement of the nymphs on four uninfected guinea-pigs, 19/24 (79%) fed nymphs from one guinea-pig and 4/25 (16%) fed nymphs from a second guinea-pig were infected; all the ticks examined from the other two guinea-pigs were uninfected. The results suggest that TBE virus was transmitted from a low proportion of infected nymphs (infected as larvae) to uninfected nymphs as they fed together on an uninfected guinea-pig. Such amplification of the initial infection, at the population level, could play an important role in maintaining TBE virus infections in nature, particularly if there is a low level of vertical transmission from one tick generation to the next.     

Immunization of cattle with a semi-purified fraction of solubilized membrane-bound antigens extracted from the midgut of the tickRhipicephalus appendiculatus

Boran cattle were immunized with a semi-purified fraction (F₂) of solubilized membrane-bound antigens extracted from the midgut of the tickRhipicephalus appendiculatus. A subsequent challenge with all stages ofR. appendiculatus showed protection of the cattle to a significant degree. There was a decrease in engorgement weight of ticks, the number which fed above critical engorgement weight was considerably reduced, and about 38% of the larvae which fed to repletion did not moult. The major cause of death of adult female ticks was the increased grooming by cattle as a result of increased dermal irritation.     

Molecular individuality and adaptation of the tick Rhipicephalus appendiculatus in changed feeding environments

The tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) naturally infests many host species. However, the mechanisms that enable it to feed on such a wide range of hosts are unclear. One possibility is that a tick population maintains molecular (genotypic and/or phenotypic) diversity among individuals such that individuals vary in their competency in taking bloodmeals under different feeding conditions. As a first step in testing this hypothesis, we showed that the polymorphism of salivary gland proteins, previously demonstrated in unfed ticks, was maintained during feeding on guinea-pigs. We then compared feeding performance under standard laboratory rearing conditions: one instar (adults or nymphs) feeding on guinea-pigs, with three changed conditions: (1) two instars (adults and nymphs) feeding together on guinea-pigs; (2) one instar (adults or nymphs) feeding on hamsters; and (3) two instars (adults and nymphs) feeding together on hamsters. The mean engorged weight of adult females was significantly reduced under all changed conditions, indicating that most of the adult individuals were significantly challenged by the changed conditions. However, some individuals achieved successful engorgement, indicating competence to the changed condition, and demonstrating variation in adaptive ability among individuals. Engorged females produced egg masses positively correlated to the engorged weights. More interestingly, the correlation coefficient (R) increased when feeding condition was changed. This may lead to more efficient selection for population adaptation under the changed conditions. As the feeding success of ixodid ticks depends on the efficiency of the cocktail of immunomodulatory saliva, the relevance of the polymorphism of salivary gland proteins and host adaptation is discussed.     

A hybridization model forRhipicephalus appendiculatus andR. zambeziensis (Acarina: Ixodidae) using glucose-6-phosphate-isomerase isoenzymes

In the Eastern Province of Zambia,Rhipicephalus appendiculatus (Neumann) andR. zambeziensis (Walker) are sympatric. Intermediate forms as well as typical specimens are found. No morphological criteria could detect cross-breeding between these species in the field.Hybrids betweenR. appendiculatus andR. zambeziensis were produced and glucose-phosphate-isomerase (EC 5.3.1.9.; GPI) isoenzymes resolved by agarose electrophoresis. Phenotyping hybrids in the F1 and F2 generations was explained by the autosomal transmission of two loci of GPI-genes. Identification of some hybrid phenotypes offers the possibility of showing presently undetected hybridization in the field. A genetic model is proposed to explain the patterns.     

Resistance in cattle againstRhipicephalus appendiculatus with an assessment of cross-resistance toR. pulchellus (Acari: Ixodidae)

A comparison of the yield of engorged nymphs ofRhipicephalus appendiculatus andR. pulchellus was carried out between cattle highly resistant toR. appendiculatus but susceptible toR. pulchellus, and cattle and rabbits susceptible to both species.Resistant cattle yielded fewer and smallerR. appendiculatus nymphs than either susceptible cattle or rabbits. More nymphs of both tick species dropped from susceptible cattle but no differences were detected in the mean weight of engorgedR. pulchellus nymphs fed onR. appendiculatus-resistant and susceptible cattle, although resistant animals yielded more nymphs ofR. pulchellus than ofR. appendiculatus.An acute inflammatory reaction, accompanied by intense pruritus and grooming, was observed in the ears of the resistant cattle infested withR. appendiculatus, but no reaction was detected in the opposite ears whereR. pulchellus was feeding.The low cross-resistance between these two closely related tick species suggests that different antigens may be involved in the development of resistance in cattle to different tick species.     

Feeding performance ofAmblyomma variegatum (Acarina: Ixodidae) fed repeatedly on rabbits

Rabbits infested with different stages ofAmblyomma variegatum Fabricius, 1794 became resistant to subsequent infestations by the same tick. Resistance was manifested by a reduction of 73.6% in the mean engorged weight of female ticks by the 3rd infestation. By the 5th infestation, only 70% of the nymphs engorged and their mean engorged weight was 57.7% of that of nymphs fed on tick-naive control rabbits. In the case of larvae, there was a 22.8% reduction in their mean engorged weight by the 6th infestation. Based on increases in body length while feeding on tick-naive rabbits, the nymphs could be divided into four feeding categories, i.e. Nl (unfed), N2, N3 and N4 (increasing states of engorgement). While 92–96% of ticks which dropped from susceptible rabbits fitted into feeding category N4, only 28% of those that dropped from tick-resistant rabbits fitted into that category. The remainder of the ticks fitted into categories N3 (60%) and N2 (12%). The majority of those ticks with reduced weight developed into adults which had an atypical scutal ornamentation pattern, in that an additional pair of lateral spots was regularly observed on the males. The females of such ticks deposited small numbers of eggs from which no larvae hatched.     

Immunological responses of the rabbit host to infestation by the brown ear-tickRhipicephalus appendiculatus (Acarina: Ixodidae)

Rabbits developed resistance toRhipicephalus appendiculatus instars following repetitive infestations. Rejection was accompanied by elevated IgM and IgG titres. Extracts of salivary gland, mouthpart cement and whole ticks induced a dose-related lymphocyte transformation.Skin-provocation tests with tick extracts elicited an immediate type-I hypersensitivity reaction with a delayed time-course which was influenced by antihistamines. Passive-transfer studies indicated that resistance was partially transferred with serum.A comparative histological study of the attachment sites of larvae on resistant and naive hosts demonstrated the role of eosinophils and macrophages during the initial phase of infestation. Possible rejection mechanisms are discussed in the light of these and other findings.     

In-vivo effects of ivermection onRhipicephalus appendiculatus: the influence of tick feeding patterns and drug pharmacokinetics

Sublethal effects seen amongstRhipicephalus appendiculatus feeding on ivermectin-treated rabbits were diverse and dependent both on drug dose, pharmacokinetics and tick feeding patterns: changes in drug formulation, the time of infestation relative to treatment, and the tick instar used, profoundly influenced acaricidal activity. Death was a sequel to paralysis only if tick feeding was interrupted for sufficient time to produce irreversible dehydration. Concurrent pharmacokinetic investigations revealed that, for the larvae ofR. appendiculatus, the mean critical lethal dose of ivermectin imbibed over a 5-day engorgement period was 3500 g/kg. This quantity of ivermectin was achieved in the blood-meals of larvae feeding on rabbits treated subcutaneously with a single dose of Ivomec injection (MSD)^*800 g/kg, provided infestation took place within 24 h of treatment. At lower drug doses, or if larval infestations were delayed for>24 h post-treatment, the quantity of circulating ivermectin (and thus imbibed by the tick larvae) fell below 3500 g/kg and an increasing percentage of larvae successfully engorged and detached. More than 90% of such larvae moulted to the nymphal stage. Nymphae and larvae exhibited similar susceptibility to ivermectin on treated rabbits which could be explained by similar feeding patterns. However, adult female and male ticks were markedly less susceptible and interpretation of ivermectin-induced effects was more complex.     

Visual Evaluation and Recognition of Hosts by the Tick Parasitoid, Ixodiphagus hookeri (Hymenoptera: Encyrtidae)

The role of host size, movement, feeding status, color, and species in the visual host evaluation and recognition behavior of the tick parasitoid, Ixodiphagus hookeri Howard was investigated. Freshly emerged female parasitoids were subjected to a choice bioassay, where the test materials were placed in sealed vials and the vials placed in a Petri dish. When presented with A. variegatum live and mummified nymphs, females examined: larger nymphs significantly longer than smaller nymphs, fed nymphs significantly longer than unfed nymphs, dead and live nymphs for similar lengths of time, and grey live nymphs and yellow-brown and dark brown mummified nymphs for similar lengths of time. The total number of visits to vials containing these test materials were also not significantly different, except there were significantly more visits to yellow-brown mummies when compared to the number of visits to dark brown mummies. When presented with A. variegatum (host) and R. appendiculatus (nonhost) nymphs, the females examined A. variegatum nymphs significantly longer than R. appendiculatus nymphs. The total number of visits to vials containing A. variegatum nymphs were significantly more than the visits to the vials containing R. appendicualtus nymphs. Furthermore, females spent significantly more total examination time per visit on larger and fed A. variegatum nymphs when compared to smaller and unfed nymphs, respectively. Direct and indirect detection were significant when females were presented with fed versus unfed A. variegatum nymphs, grey nymphs versus yellow-brown mummies, and R. appendiculatus versus A. variegatum nymphs. Direct and indirect detection for the rest of the bioassays were not significantly different. Finally, The percentages of females contacting large fed A. variegatum nymphs first were significantly different from those of females contacting small unfed R. appendiculatus nymphs first. The firstcontact percentages for the rest of the bioassays were not significantly different.     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.