Factors influencing infections in Rhipicephalus appendiculatus ticks fed on cattle infected with Theileria parva.

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.

Optimization of the in vitro feeding of Rhipicephalus appendiculatus nymphae for the transmission of Theileria parva.

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 degrees 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.

Estimation of heritability of susceptibility to infection with Theileria parva in the tick Rhipicephalus appendiculatus.

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.

Transmission of Theileria parva to cattle by Rhipicephalus appendiculatus adults fed as nymphae in vitro on infected blood through an artificial membrane.

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 piroplasma 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.

In vitro feeding of instars of the ixodid tick Amblyomma variegatum on skin membranes and its application to the transmission of Theileria mutans and Cowdria ruminatium.

An in vitro feeding method using rabbit or cattle skin membranes, applied successfully to all stages (larvae, nymphae and adults) of the ioxodid tick, Amblyomma variegatum, is described. The feeding apparatus consisted of a blood container with a membrane placed on top of a tick containment unit. A carbon dioxide atmosphere of between 5 and 10% and a temperature of 37 degrees C were used as stimulants for the attachment of the ticks. High CO2 concentrations in the atmosphere improved the feeding success of all instars. The effect of anticoagulation methods for the bloodmeal was investigated, and heparinized blood was found to be the most suitable for tick feeding. When the bloodmeal was replaced by tissue culture medium for feeding nymphs the subsequent moulting success was reduced. Adult ticks of both sexes remained attached for up to 16 days, until completion of their bloodmeals. All stages of the tick fed on whole blood in the artificial feeding system and all reached engorged weights less than those achieved by control ticks fed on experimental animals. A large proportion of ticks, fed artificially on whole blood, moulted or laid eggs successfully. The method was successfully applied for the transmission of Theileria mutans and Cowdria ruminantium to cattle.