Lumpy skin disease (LSD) is an economically important, acute or sub-acute, viral disease of cattle that occurs across Africa and in the Middle East. The aim of this study was to investigate if lumpy skin disease virus (LSDV) can be transmitted mechanically by African brown ear ticks (Rhipicephalus appendiculatus Neum.). Laboratory-bred R. appendiculatus males were fed on experimentally infected viraemic ‘donor’ cattle. Partially fed male ticks were then transferred to feed on an uninfected ‘recipient’ cow. The recipient animal became viraemic, showed mild clinical signs of LSD and seroconverted. Additionally, R. appendiculatus males were found to transmit LSDV through feeding on skin lacking visible lesions, demonstrating that viraemic animals without lesions at the feeding site of ticks may be a source of infection. This is the first time that transmission of poxviruses by a tick species has been demonstrated and the importance of this mode of transmission in the spread of LSDV in endemic settings is discussed.

The initiation of feeding of infected Rhipicephalus appendiculatus adults induces the rapid development of Theileria parva sporoblasts within the salivary gland acini leading to the production of numerous sporozoites which are inoculated into the mammalian host initiating infection. In this study the pattern of development, host cell specificity and emission of T. parva sporozoites within the salivary glands of heavily infected, 4-day fed adult R. appendiculatus ticks was examined. Infected acini were randomly distributed throughout the salivary gland. Sporozoite development within each gland was not synchronized and wide variation in the rate of parasite development, which correlated with the secretory activity of the individual acinus, was observed in all glands examined. Previous studies had shown that T. parva developed primarily in Type III ‘e’ cells. However, in heavily infected salivary glands sporogony and the emission of mature sporozoites also occurred in ‘c’ cells of Type II acini. Sporozoite emission from infected cells occurred by a process similar to apocrine secretion. The loss of the apical membrane of the infected cell allowed sporozoites free access to the lumen of the acinus and into the collecting ducts of the salivary gland. Sporozoite discharge was gradual since few parasites were found in the acinus valve or in the collecting ducts. Furthermore, the small size of the acinar valve aperature ensures that only small numbers of sporozoites can be released at any one time from an infected acinus.

A large database on the transmission of a stabilate of the Theileria parva Muguga stock from one breed of cattle using two stocks of Rhipicephalus appendiculatus, Muguga and Ol Pejeta was developed and analysed. Factors associated with the ticks and cattle, and the infections developing in cattle were studied in relation to the infection variables in the tick batches harvested daily from cattle. Generalized Linear Interactive Modelling (GLIM) was used to determine the importance of factors and interactions in influencing the levels of tick infection variables using Type I and Type III sums of squares analyses. Analysis of the 6 variables, prevalence (percentage of ticks infected), abundance (mean number of infected salivary gland acini per tick examined) and intensity (mean number of infected salivary gland acini per infected tick) in batches of 30 male and 30 female ticks showed that 24 covariates, factors or interactions had a significant effect (P> 0·05). Certain covariates and factors were particularly important for all 6 tick infection variables; parasitaemia of animal on the day of tick harvest, stabilate dilution administered to animal, month in which tick batch was harvested, minimum packed cell volume of animal over the sampling period, age of animal, and the minimum leukocyte count of the animal over the sampling period. The GLIM analyses were found to be a useful tool in identifying factors that influence infection levels and in devising methods of producing tick batches with more predictable infections.

Heritability of susceptibility to infection with Theileria parva was estimated from full sib families of Rhipicephalus appendiculatus ticks. Male and female ticks of 2 stocks were mated singly. Nineteen full sib families of the Muguga stock and 17 full sib families of the Kiambu stock were obtained. Nymphae of these families were fed on cattle infected with T. parva so that the ticks became replete on days 16 and 17 after infection when the blood was parasitaemic with intraerythrocytic piroplasms. The T. parva infections were assessed in the resultant adult ticks of each full sib group and the abundance of infection, the number of salivary gland acini infected/tick, was found to be the most useful parameter for analysis. Estimates of heritability of the susceptibility to infection with T. parva for the Kiambu and the Muguga tick stocks were 0·24 and 0·26 respectively. Using only the data from ticks which fed on day 16, the heritability estimates were 0·39 for the Kiambu stock and 0·59 for the Muguga stock. These results indicate that tick lines of high or low susceptibility for T. parva infection could be produced through selection.

A guinea-pig immunoglobulin-G linked agarose column was used to detect the immunoglobulin-G binding proteins (IGBP) in the ixodid ticks, Rhipicephalus appendiculatus, Amblyomma variegatum and Ixodes hexagonus. IGBPs were found in salivary gland extract (SGE) of the unfed ticks of all 3 species. The sizes of the IGBPs, as determined by SDS–polyacrylamide gel electrophoresis, differed between species. In R. appendiculatus 2 IGBP bands were detected in haemolymph collected from unfed females and males, and an additional 78 kDa IGBP was detected in haemolymph samples of male and female ticks that had fed for 6 days. Although there was no difference in the IGBP profile of SGE derived from unfed male and female R. appendiculatus, 2 new IGBPs were detected in the SGE of the male ticks after they had fed for 6 days. The possible function of IGBP in ticks is discussed.

To compare the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks, antisera were prepared from guinea-pigs immunized with soluble denatured salivary gland extracts (SGE). The extracts were derived from R. appendiculatus female ticks that were either unfed (day 0) or partly fed (day 6). The sera were used in immunoblotting, following SDS–polyacrylamide gel electrophoresis, to examine the antigen profiles during the course of tick feeding on guinea-pigs. Day 0 and day 6 SGE antisera appeared to detect common proteins in the different tick samples. For example, haemolymph apparently shared some of the small protein bands (31·5–34 kDa) detected in SGEs. These small proteins appeared in both samples at the same stage of feeding, suggesting that haemolymph and salivary glands not only have common antigens but may also share some functions. Furthermore, a number of protein bands were detected in haemolymph before they were apparent in the salivary glands or saliva. Thus some antigens detected in the salivary glands and saliva may be derived from the haemolymph. The results indicate that the host may be exposed to tick saliva antigens that are also present in the haemolymph. We discuss the significance of these observations with regard to the induction of host immunity to ticks and the development of tick vaccines.

Groups of cattle were immunized with 10−2 dilutions of sporozoite stabilates of Theileria parva lawrencei derived from African buffaloes either alone or in combination with Theileria parva parva derived from cattle and concomitant treatment with either long or short-acting formulations of oxytetracyline. At 90 or 120 days after infection, uninfected Rhipicephalus appendiculatus nymphal ticks were applied to individual immunized cattle and the resultant adults ticks were applied to individual susceptible cattle. Theilerial infection developed from ticks fed on 6 out of 11 animals investigated for evidence of a carrier state. Two additional animals were shown by cell-culture isolation to have persistent theilerial infections. Nine cattle infected with the parasites from carrier animals were treated with parvaquone and 7 recovered. These recovered cattle were then challenged with the original immunizing stabilates at 10° dilution together with the original immunized and carrier cattle. Six out of 7 cattle which had recovered from carrier-derived infection succumbed to this challenge and died but none of the original immunized cattle showed theilerial reactions. When a carrier-derived sporozoite stabilate was used to challenge cattle immune to the original immunizing parasite, they proved to be immune. Cattle immune to the carrier-derived parasites were all immune to challenge with the original parasite. A monoclonal antibody profile aginst T. parva schizonts isolated by cell culture from samples of the experimental animals did not appear to be sensitive enough to determine the antigenic differences between the carrier-derived parasite and the original immunizing parasite. Indications are that the carrier state is not likely to produce new antigenic strains which would be dangerous to immunized cattle.

An apparatus for artificial feeding of Rhipicephalus appendiculatus nymphae was modified to improve feeding performance. Heparinized blood was supplied above a treated artificial membrane while the ticks attached below on its undersurface. The feeding apparatus was incubated at 37 °C in an atmosphere of 3% CO2 concentration and a relative humidity of 75–80%. Under these conditions, 91% of the engorged nymphae attained a mean weight of 6–11 mg, and an average of 93% of those nymphae moulted into adults. When this system was used to feed nymphal ticks on blood infected with Theileria parva piroplasms, the mean prevalence of infection in the resultant female and male ticks was 86% and 54%, respectively. The feeding performance and T. parva infection levels were comparable to those of nymphal ticks fed on the blood donor cattle. The apparatus used in this study has potential for modification to suit the artificial feeding needs of other species of ixodid ticks and for use in investigations to examine other tick/pathogen relationships.

A comparison of feeding and breeding performance was made between a laboratory strain (LS) and two freshly collected field strains (FS1, FS2) of Rhipicephalus appendiculatus. The LS had been cultured for over 30 years on tick-susceptible rabbits. When fed on susceptible rabbits LS produced smaller eggs and unfed larvae, nymphs and adults than those of FS. Eggs and larvae from females engorged on cattle were larger than those from females engorged on rabbits. LS and FS females engorged on susceptible hosts were of similar weight, but the FS females were twice as heavy as LS females when tick-resistant hosts were used. A higher proportion of LS ticks than of FS ticks engorged on susceptible hosts, but a lower proportion on resistant hosts. Thus on susceptible hosts LS showed a higher reproduction efficiency than FS, but a lower one than FS on resistant hosts. The proportional reductions of engorged weights and numbers feeding successfully on resistant hosts were significantly lower for LS than FS ticks. Cross-resistance was low between LS and FS. Although all strains were affected by host resistance, it was concluded that the field strains were better adapted to withstand resistance than the laboratory strain.

Host immunoglobulin G (IgG) crossed the gut wall into the haemocoel of adult Rhipicephalus appendiculatus female ticks when they fed on guinea-pigs. Guinea-pig IgG was also found in saliva of the feeding ticks. The concentration and antibody activity of IgG in haemolymph, salivary gland extract (SGE) and saliva at different stages of tick feeding were detected by enzyme-linked immunoassay. Specific activity of the IgG in tick samples was determined by feeding ticks on guinea-pigs which were immunized with killed Escherichia coli: 35–42% of the antibody activity in guinea-pig immune serum remained in the tick samples. The high relative concentration of IgG in tick saliva at later stages of feeding suggests that the tick may have a mechanism for getting rid of foreign proteins via the salivary gland. Such a mechanism could involve lgG binding proteins (1GBPs) which were found in both haemolymph and SGE of female ticks at day 6 of feeding using a guinea-pig IgG–agarose affinity column. In female ticks, the Mr of 1GBPs in SGE (23 and 57 kDa) were less than those in haemolymph (78 and > 100 kDa). The existence of 1GBPs in both the tick salivary gland and haemolymph indicate that haemolymph and salivary gland cooperate to remove foreign proteins, e.g. host immunoglobulin, from the body during feeding. This mechanism may be a part of the tick self-defence system.

The transmission of Theileria parva by nymphal and adult Rhipicephalus appendiculatus was compared by the assessment of salivary gland infections in tick batches fed on the same group of infected cattle at the same time. When larval and nymphal R. appendiculatus Muguga ticks were fed concurrently on cattle undergoing acute infection with T. parva Muguga, the resultant nymphae developed a slightly lower prevalence of infection than did the adult ticks. The abundance of infection was 5–20 times higher in the adult ticks than in the nymphae. When larval and nymphal R. appendiculatus Muguga and R. appendiculatus McIlwaine were fed to repletion on cattle infected with T. parva Boleni, a parasite causing subacute infection, resultant adult tick batches had a relatively high prevalence of infection, but infection was not detected in resultant nymphal batches. When cattle that were carriers of 2 stocks of T. parva, Marikebuni and Kiambu 5, were used as the source of infection, the infections developing in adult R. appendiculatus Muguga ticks were much higher than those developing in nymphae. The structure of salivary glands differed between nymphal ticks, adult males and adult females, and this is considered to be an important factor affecting the infection levels. The morphology of the type III acini, the target acini for sporogony, was similar, but the mean numbers of type III acini were different, with 87 in nymphae, 1346 in males and 1736 in females. This difference was correlated with the different infection levels produced in the various instars and sexes. While the process of sporogony in the different tick instars and sexes was similar, the rate of sporogony was fastest in feeding nymphae, taking on average 2–3 days, compared to 3–4 days in females and an irregular period in the males. These results are discussed in relation to the epidemiology of T. parva.

A technique is described for the efficient feeding of Rhipicephalus appendiculatus nymphae on cattle blood through an artificial membrane bearing tactile and olfactory stimuli. The effect of four anticoagulation methods on the feeding of nymphae was compared and heparinized blood was found to be the most efficacious, followed by defibrinated blood. Blood treated with acid citrate dextrose (ACD) or ethylenediamine tetraacetate (EDTA) inhibited nymphal feeding. Nymphae fed on heparinized and defibrinated blood obtained engorgement weights within the range of ticks fed on mammalian hosts and they subsequently moulted and fed normally as adults and produced viable eggs. Nymphae fed on membranes using either defibrinated or heparinized blood infected with Theileria parva piroplasms developed salivary gland infections as adult ticks and transmitted East Coast fever (ECF) to susceptible cattle. There were indications that T. parva-infected defibrinated blood was not as infective to the feeding nymphae as the infected heparinized blood. When T. parva-infected heparinized blood was used to feed nymphae through membranes in two experiments, it was found that the infections in the resultant adult ticks could be comparable to those of nymphae fed on donor cattle, but were usually lower. The membrane feeding technique will enable the study of factors affecting the tick and T. parva transmission without the complication of host/T. parva interactions and could be useful for both tick maintenance and Theileria parasite isolation and maintenance.

Five groups of adult Rhipicephalus appendiculatus Neumann, with an average of 98 Theileria parva-infected salivary gland acini per tick, were exposed to four different temperature conditions in the laboratory or to quasi-natural conditions in Kenya. The survival of the infected ticks and T. parva parasites in their salivary glands were determined over time. Ticks kept under quasi-natural conditions survived up to 86 weeks. This period was longer than that of ticks exposed to any of the four laboratory conditions, except the diurnal temperature rhythm of 13–23 °C and 85% relative humidity (RH) to which it was comparable. Theileria parva survived for 82 weeks in ticks exposed to quasi-natural conditions, a period which was much longer than that of the parasites in ticks maintained under any of the laboratory conditions. Survival of the infected ticks and their infections seemed to be adversely affected by higher temperatures in the laboratory or drought in the field. There was an apparent density-dependent influence in parasite survival, with a dramatic fall in infection occurring in the more highly infected ticks before stabilizing at lower levels.

Groups of nymphal Rhipicephalus appendiculatus Muguga, having a mean of 1 or 9 Theileria parva Muguga-infected salivary gland acini per tick, were kept under quasi-natural conditions at an altitude of 1950 m or 20°C at a relative humidity of 85% in the laboratory and their survival and infection prevalence and abundance determined over time. Theileria parva infections for both categories of ticks survived in the nymphal ticks for 50 or 26 weeks post salivary gland infection under quasi-natural or laboratory conditions respectively. There was a distinct decline in infections in the more heavily infected nymphae under both conditions of exposure, reflecting an apparent density dependence in parasite survival. Nymphal ticks having an average infection level of 1 infected salivary gland acinus per tick, survived for up to 69 or 65 weeks post-repletion under quasi-natural or the laboratory conditions respectively. Nymphae having an average infection level of 9 infected salivary gland acini per tick survived for a similar duration under each of the 2 conditions. The infection level of 9 infected salivary gland acini per tick did not seem to significantly affect the survival of the tick vector compared to those having an average of 1 infected salivary gland acinus per tick.

Gregariousness can be advantageous in interspecific competition while intraspecific competition may favour solitude. We examined feeding behaviour of the ixodid tick, Rhipicephalus appendiculatus, in the context of interspecific (tick–host) and intraspecific (tick–tick) competition. Such competition is mediated through host rejection responses to tick infestation to which ticks respond by secreting immunodulatory saliva. We observed that group feeding adults increased their blood-feeding rate, reducing the time to mating and repletion, compared with individual feeding of paired adults. The benefits of feeding aggregation indicate direct reciprocity between ticks, most likely resulting from the shared activities of their bioactive saliva. However, fast-feeding ticks appeared to impair blood-feeding success of slow-feeding females during group feeding. This may be explained by the faster feeders exacerbating host responses on detachment that are then directed against the slower feeders. As female fecundity is generally proportional to the size of the bloodmeal, there will be a selection pressure to feed gregariously. Greater understanding of the benefits and costs of feeding aggregation may help to improve tick control strategies.

Haematophagous insect vectors of arthropod-borne viruses (arboviruses) feed repeatedly. Consequently, they can transmit arboviruses to more than one host during the same developmental stage (intra-stadial transmission). By contrast, ixodid ticks generally feed only once at each parasitic stage (larva, nymph, and adult) and hence they have only one opportunity for tick-borne virus transmission per stadium (inter-stadial transmission). Under natural conditions, tick-infested hosts may die (from disease or other causes) before the ticks have completed their long period of engorgement. A laboratory model was used to investigate the consequences of premature host death on tick-borne virus transmission. We report intra-stadial transmission of Thogoto virus by the nymphal, male, and female ticks of Rhipicephalus appendiculatus. Tick-borne Thogoto virus infection caused viraemia and death of hamsters before the nymphal and adult ticks feeding on them had completed engorgement. The resulting partially fed ticks were allowed to continue engorgement on new, uninfected hosts (interrupted feeding). During feeding on the subsequent hosts, they transmitted the virus intra-stadially to susceptible hosts (hamsters), and to uninfected co-feeding ticks on non-susceptible hosts (guinea-pigs). Intra-stadial transmission, mediated by interrupted feeding, may help explain outbreaks of rapid and fatal tick-borne viral diseases, and the epidemiology as well as evolution of virulence, in a susceptible host population. Additionally, intra-stadial transmission provides an increased risk of tick-borne pathogen transmission to humans and domestic animals during slaughter and game hunting.

Two-dimensional gel electrophoresis was used to compare the protein profiles of two geographically isolated populations of the tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) in Kenya. Most of the protein spots were common to both populations, but a number were specific to each population. Since proteins are encoded by genes, the presence of population-specific proteins suggests that there may be genetic differences between the two populations. It is proposed that some of these population-specific proteins might be related to the differences in susceptibility of the ticks to Tlwileria pnrva infection.

Ixodid female ticks take one comparatively large bloodmeal which they convert to a single large egg mass and then they die. To examine the outcome of interrupted feeding, equal numbers of male and female Rhipicephalus appendiculatus adult ticks were fed on guinea pigs (host 1) for either 2, 4, or 6 days, or to engorgement (8 days). All of the fully engorged (D8) females laid a single large egg mass (80–160 mg/tick), while 85% of the day 6-fed (D6) female ticks (n=20) each laid a small egg mass (6·1 mg/tick). None of the females that had fed for 2 or 4 days oviposited. Ninety percent (n=20) of the day 2-fed (D2) females survived for 4 weeks after their feeding was interrupted, whereas 65% (n=20) of the day 4-fed (D4) females survived. All of the surviving partially fed female ticks (D2 and D4) attached to a second guinea pig (host 2) and attained engorged body weights that were not significantly different from those of the control females (P<0·05). Female ticks that engorged following interrupted feeding layed egg masses comparable to the controls, indicating that engorgement on host 2 was successful. The salivary gland protein profile of female ticks changed constantly during feeding. However, when feeding was interrupted, the protein expression pattern switched back to that of the non-parasitic state, presumably to enable the partially fed ticks to survive and reattach on the new host. This observation indicates that female ixodid ticks have a natural ability to survive and re-establish successful feeding on a new host if the first attempt at feeding is unsuccessful. Such an interrupted feeding mechanism supports the hypothesis that partially engorged ticks may play a role in tick-borne pathogen transmission.