The Rhipicephalus appendiculatus tick vector of Theileria parva is absent from cape buffalo (Syncerus caffer) populations and associated ecosystems in northern Uganda.

Rhipicephalus appendiculatus is the major tick vector of Theileria parva, an apicomplexan protozoan parasite that causes the most economically important and lethal disease of cattle in East and central Africa. The African cape buffalo (Syncerus caffer) is the major wildlife host of T. parva from southern Uganda and Kenya to southern Africa. We show herein that R. appendiculatus appears to be absent from the two largest national parks in northern Uganda. Syncerus caffer is common in both of these national parks, specifically Murchison falls (MFNP) and Kidepo Valley (KVNP). We re-confirmed the previously reported absence of T. parva in buffalo sampled in the two northern parks based on RLB data using a nested PCR based on the T. parva p104 gene. By contrast, T. parva-infected R. appendiculatus ticks and parasite-infected buffalo were present in Lake Mburo (LMNP) in South central Uganda. This suggests that the distribution of R. appendiculatus, which is predicted to include the higher rainfall regions of northern Uganda, may be limited by additional, as yet unknown factors.

Differential transcription of two highly divergent gut-expressed Bm86 antigen gene homologues in the tick Rhipicephalus appendiculatus (Acari: Ixodida).

The transcriptional control of gene expression is not well documented in the Arthropoda. We describe transcriptional analysis of two exceptionally divergent homologues (Ra86) of the Bm86 gut antigen from Rhipicephalus appendiculatus. Bm86 forms the basis of a commercial vaccine for the control of Rhipicephalus (Boophilus) microplus. The R. appendiculatus Ra86 proteins contain 654 and 693 amino acids, with only 80% amino acid sequence identity. Reverse-transcription PCR of gut cDNA showed transcription of only one genotype in individual female ticks. PCR amplification of 3' untranslated sequences from genomic DNA indicated that both variants could be encoded within a single genome. When both variants were present, one of the two Ra86 genotypes was transcriptionally dominant.

The persistence of Theileria parva infection in cattle immunized using two stocks which differ in their ability to induce a carrier state: analysis using a novel blood spot PCR assay.

An improved Theileria parva DNA detection assay based on the polymerase chain reaction (PCR) using primers derived from the 104 kDa antigen (p104) gene was developed to detect parasite DNA in blood spots on filter paper. The specificity of the assay was validated using DNA from a wide range of cattle-derived and buffalo-derived stocks of T. parva. DNA of T. annulata, T. buffeli, T. lestoquardi, T. mutans and T. taurotragi was not amplified using the p104 primers. The detection threshold of the assay was approximately 1-2 parasites/microl of infected blood. PCR amplification using the p104 primers was applied to sequential samples from groups of cattle experimentally infected with either the T. parva Marikebuni stock that induces a long-term carrier state or the Muguga stock, which does not induce a carrier state. The study extended for up to 487 days post-infection and PCR data from defined time points were compared with parasitological microscopy and serological data, together with xenodiagnosis by experimental application of ticks. Microscopy first detected piroplasms between days 13 and 16 after infection whereas all cattle became PCR +ve between days 9 and 13. Animals infected with the Muguga stock of T. parva had parasite DNA in the peripheral blood, which could be detected by PCR, for between 33 and 129 days post-infection in different animals. By contrast parasite DNA in the blood of cattle infected with the Marikebuni stock could be detected consistently from day 9 up to 487 days, when the study terminated. The data suggest that the nature and persistence of the carrier state may differ markedly between different T. parva parasite stocks.

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.

Detection of Theileria parva in the salivary glands of Rhipicephalus appendiculatus: evaluation of staining methods.

A comparison of ten methods for staining tick salivary glands for detection of Theileria parva infection from ticks fed on rabbits for various periods was undertaken. Staining with azure without hydrochloric acid hydrolysis was found to be the most reliable method for detection of the presporozoite stages (sporoblasts) of T. parva in the salivary gland acini of unfed Rhipicephalus appendiculatus and could be applied to field ticks. All the stains proved suitable for the detection and quantitation of sporozoites in ticks fed for 4 days on rabbits. The capacity of the stains to allow detection of early stages of T. parva differed, but it became more reliable during tick feeding as sporoblasts developed and matured. Giemsa's stain and Feulgen's stain followed by superimposition of Giemsa's stain were superior to other stains for the detection and quantitation of immature salivary gland stages in feeding ticks.

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.

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.

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.