Parasitol Res (1995) 81:74-81 Springer Verlag 1995

W. R Voigt S. N. Mwaura G. M. Njihia S. G. Nyaga A. S. Young Detection of parva in the salivary glands of Rhipicephalu$ appendiculatu, evaluation of staining methods

Received: 15 April 1994 / Accepted: 15 July 1994

Abstract A comparison of ten methods for staining of the parasite. The cycle of T. parva in the tick is con- salivary glands for detection of Theileria parva infection cluded by sporogony in the salivary-gland acinar cells of from fed on rabbits for various periods was under- nymphal or adult ticks, usually type III acinar cell "e" taken. Staining with azure without hydrochloric acid hy- (Fawcett et al. 1982, 1985), when the sporozoites are drolysis was found to be the most reliable method for de- emitted from the salivary-gland acinus in the saliva into tection of the presporozoite stages (sporoblasts) of T. the mammalian host during feeding. The ability to detect parva in the salivary gland acini of unfed Rhipicephalus Theileria parasites reliably in salivary glands and to as- appendiculatus and could be applied to field ticks. All sess infection levels is important in studies of the the stains proved suitable for the detection and quantitat- epidemiology of theileriosis, since these parameters re- ion of sporozoites in ticks fed for 4 days on rabbits. The present the most relevant measure of the infective para- capacity of the stains to allow detection of early stages of site burden of ticks and the likely risk to cattle. T. parva differed, but it became more reliable during tick There are several methods of visualizing T. parva stag- feeding as sporoblasts developed and matured. Giemsa's es in the salivary glands of R. appendiculatus. Early stud- stain and Feulgen's stain followed by superimposition of ies used serial histological sections of the salivary glands Giemsa's stain were superior to other stains for the de- that were stained with Romanowsky stains (Cowdry and tection and quantitation of immature salivary gland stag- Ham 1932; Martin et al. 1964; Purnell and Joyner 1968), es in feeding ticks. but these were laborious and time-consuming methods. More recently, routine assessment of salivary-gland infec- tion has been obtained following staining of whole sali- Introduction vary glands with undamaged acini using the methyl green/pyronin stain (MGP; Walker et al. 1979; Irvin et al. East Coast fever (ECF) is a major cattle disease in East 1981) or Feulgen's stain (Blewett and Branagan 1973; and Central Africa caused by the protozoan parasite The- Young and Leitch 1981; Bfischer and Otim 1986). The ileria parva and is transmitted by the three-host ixodid quantitative assessment of infection with these stains usu- tick RhipicephaIus appendiculatus. The distribution of ally requires tick dissection to remove the salivary gland. the tick vector is wider than that of the pathogen (Less- Intermediately to heavily infected salivary glands can be ard et al. 1990) and, apart from transmitting T. parva, recognized under the dissecting microscope without stain- this tick is also the vector of a less pathogenic species, T. ing because of the changes in the morphology of infected taurotragi, and possibly other as yet unidentified The- acini cells. A more reliable method is to use interference ileria species. contrast microscopy to examine freshly dissected salivary T. parva is transmitted transstadially following infec- glands (Young et al. 1983), which allows the selection of tion of larvae or nymphae by feeding on cattle or buffalo infected acinar cells containing viable parasites in the sali- whose erythrocytes are infected with the piroplasm stage vary glands. If viable sporozoites are not required, the sal- ivary glands can be fixed in situ using 2.5% glutaralde- W. R Voigt1 S. N. Mwaura G.. Njihia - S. G. Nyaga hyde (unpublished results) to prevent bursting of infected A. S. Young (~) acini during examination. Staining of T. parva stages in International Laboratoryfor Research on Animal Diseases, P. O. Box 30709, crushed salivary-gland acini can be achieved using Gi- Nairobi, Kenya emsa's stain (Young et al. 1980), but quantitation is not Present address: possible with this method. New nucleic acid techniques 1Institut ftir Veterin~irmedizindes Bundesgesundheitsamts, are becoming available to detect and distinguish between Postfach 330013, 1000 Berlin 33, Germany Theileria species, which is particularly important epide- 75 miologically in the cases of T. parva and T. taurotragi spread in a single layer on a microscope slide (Bfischer and Otim (Chen et al. 1990; Bishop et al. 1994). Also, highly sensi- 1986). Three types of ticks were examined: unfed ticks, ticks that tive techniques such as the polymerase chain reaction can had been attached to rabbits for 1-3 days, and ticks that had been attached for 4 days. The glands of up to ten ticks were spread sep- be used to detect DNA of a particular Theileria species in arately on each slide. For comparison of the sensitivity of various ticks (Bishop et al. 1992; de Kok et al. 1993). stains in the detection of salivary-gland infection, each pair of sali- All available quantitative methods require some de- vary glands was separated into its two halves, and each half was gree of feeding of ticks on laboratory hosts before the in- spread on a different slide. The left side of the gland was stained with azure (without hydrolysis), whereas the right gland was fected salivary-gland acini can be stained satisfactorily stained with either MGP or Feulgen's stain. (Young and Leitch 1981). It is therefore impossible to carry out investigations directly in the field since it is necessary to transport live ticks to the laboratory and ap- Staining methods ply them to experimental animals. In many cases, ticks The stains examined were designated stains 1-10 and were subdi- detached from field hosts do not survive very long or vided according to whether they required hydrolysis (1-6) or not will not recommence feeding on a new host in the labo- (7-10). ratory (Young and Newson 1973). In this paper, ten methods are evaluated for their capaci- Stains with hydrolysis ty to stain Theileria sporozoites and earlier stages in whole salivary-gland acini following dissection and removal of For stain l, the glands were fixed in 96% methanol or ethanol for the glands from ticks fed for different periods on rabbits. 5 rain, air-dried thoroughly, and then hydrolyzed for 45-60 rain in 5 N hydrochloric acid at room temperature. The excess acid was removed by quick blotting on a filter paper (Whatman number 1), followed immediately by staining. Stain 2 was fixed and hydro- Materials and methods lyzed as described above, washed immediately after the first stain, and counterstained with the second stain. Stains 3-6 were fixed Rabbits and hydrolyzed as described above except that after hydrolysis the slides were washed under running tap water, followed immediately The rabbits used were from the ILRAD Small Animal Unit. They by staining. Stains 1-6 included the following: were maintained on commercial, coccidiostat-free guinea pig pel- 1. Feulgen's stain lets and water ad libitum and were kept at room temperature Feulgen's staining method was used as described by Btischer and (about 24 ~ C) and a relative humidity (RH) of approximately 80%. Otim (1986). Rabbits were used only once and ticks were applied in cloth bags to the ears. Ticks were removed after 1, 2, 3, or 4 days of feeding, 2. Feulgen/Giemsa's stain depending upon the experimental design. Feulgen/Giemsa's staining incorporated Feulgen's stain as de- scribed above and additional staining with 10% Giemsa's stain so- lution (Merck, Darmstadt, Germany) diluted with PBS at pH 7.4 Cattle for 2-3 min. The slides were then washed under tap water and air- dried. Boran (Bos indicus) cattle that were free of antibodies to Theileria 3. Giemsa's stain parva as assessed by the indirect fluorescent test with schizont an- The glands were stained for 5-8 rain in a 10% Giemsa's stain so- tigen (Goddeeris et al. 1982) and had been kept free of ticks be- lution diluted with PBS at pH 7.4, washed under tap water, and fore the experiments were used. air-dried. 4. Toluidine blue stain Theileria parva The gIands were stained for 3 rain in a 0.5% aqueous toluidine blue solution (Aldrich Chemical Co. Inc., Milwaukee, USA; dye A stabilate (1004) of the T. parva Muguga stock was used to infect content, 57%), washed under tap water, and air-dried. cattle. This stock has been well characterized elsewhere 5. Azure stain (Brocklesby et al. 1961; Dolan et al. 1984). The glands were stained for 30 s in a 0.1-1% aqueous azure I so- lution (BDH Chemicals Ltd., Poole, England), washed under tap water, and air-dried. Ticks 6. Methylene blue stain The slides were stained for 30 s in a 1:10 methylene blue solution Rhipicephalus appendiculatus of the Muguga stock from the IL- (Aldrich Chemical Co. Inc., Milwaukee, USA), washed under tap RAD colony were used. They originated from the East African Vet- water, and air-dried. erinary Research Organisation (EAYRO), now the Kenya Agricultur- al Research Institute at Muguga, Kenya, mad have been maintained in the laboratory for at least 40 years (Bailey 1960). Ticks were main- tained on rabbits and cattle. To obtain infected ticks, nymphal ticks Stains without hydrolysis were allowed to attach and engorge to repletion on cattle that were parasitemic for T. parva piroplasms. They moulted to adults during incubation at 24~176 and 85%-90% RH. The resultant adult Stains not requiring hydrolysis (stains 7-10) included the follow- ticks were used for staining and infection-level estimations. Unin- ing: fected ticks were maintained on rabbits to act as controls. 7. Methyl green/pyronin stain I The glands were fixed in Carnoy's fixative for 5 rain, followed by a short rinse with 70% ethanol and with distilled water. They were Preparation of slides then stained with methyl green/pyronin I (MGP I; Walker et al. 1979; Irvin et al. 1981) for 8 rain, washed under tap water, air- At 6 weeks after moulting, the salivary glands of ticks were dis- dried, and then mounted with Depex mountant (Serva Chemicals, sected out in phosphate-buffered saline (PBS) at pH 7.2 and Heidelberg, Germany). 76 8. Methyl green/pyronin stain II difference in Z parva infection between the right and heft parts of The glands were fixed for 5 rain in absolute ethanol or Carnoy's the glands using different stains. fixative, air-dried, and then stained in methyl green/pyronin II (MGP II, G. Biischer, personal communication; aqueous methyl green 0.04% and aqueous pyronin Y 0.02% in 0.5 M acetate buf- fer, pH 4.8) for 10 rain. They were then blotted with filter paper, Results differentiated in n-butanol for 3 rain, and air-dried. 9. Azure stain The glands were fixed in absolute methanol (ethanol, acetone, and Staining of salivary glands of unfed ticks infected with Carnoy's solution were also used successfully, but each fixative in- Theileria parva fluenced the result of staining in a different way) for 2-5 rain. The slides were then air-dried and stained for 30 s to 1 rain with a 0.1% (pH 4.4)-1% (pH 2.6) watery solution of azure I or other The most suitable stain for detection of Theileria-infect- forms of azure. The slides were then differentiated for 30 s to 1 ed salivary gland acini in unfed ticks was found to be rain at 15 s intervals in n-butanol. azure stain without hydrolysis. The Theileria parasites 10. Methylene blue stain appeared to be fairly densely stained, showing distinctly The glands were fixed in absolute methanol (ethanol, acetone, and delineated blue to purple areas in which other structures Carnoy's solution were also used successfully, but each influenced were not visible (Fig. 1A). The nuclei of infected cells the staining characteristics) for 2-5 rain and air-dried. They were then stained for 30 s to 1 min with a 0,1% (pH 6.3) - 1% (pH 3.8) were usually not visible. The other stains that allowed aqueous solution of methylene blue (Aldrich Chemical Co. Inc., the detection of Theileria parasites in unfed ticks were Milwaukee, USA) and washed gently under tap water. Giemsa's, FeutgerdGiemsa's and toluidine blue stain as well as azure stain with hydrolysis and methylene blue stain with hydrolysis. Neither Feulgen's stain nor MGP Examination of stained slides was useful in staining Theileria parasites in unfed ticks. Examination of stained slides was carried out under low magnifi- With Giemsa's stain the Theileria parasites appeared cation (100 X). The morphology of infected acini was determined as densely staining violet-blue granules that usually cov- by comparing these with control salivary-gland preparations from ered at least half of the acinar area. The nuclei of infect- uninfected ticks and by their comparison with the other half-gland ed cells were enlarged and distinct but stained the same stained with standard MGP I or Feulgen's stain. The main criteria for identification were the staining of parasite masses, the staining color as the nuclei of uninfected cells. The cytoplasm of of the enlarged acinar nucleus, and, where applicable, the in- uninfected cells had pink to orange cytoplasm. A combi- creased size of infected acinar cells. When necessary, the magnifi- nation of Giemsa's and Feulgen's stain provided a stain- cation was increased to 400 X for clarification of these criteria. ing quality similar to that of Giemsa's stain but gave a The results of staining the two halves of the salivary glands were analyzed using the Mann-Whitney nonparametric test or greater differentiation of infected acini (Fig, 1B). analysis of variance (ANOVA test, nested design, 5% significance Toloudine blue staining with hydrolysis allowed the level; Steel and Torrie 1980) to test whether there was a significant identification of parasites in unfed ticks, and the The-

Fig. 1A, B Staining of salivary-gland acini of unfed Rhipicephalus appendiculatus infected with Theileria parva (magnification, • The in- fected acinar cells are indicated with arrows. A Azure stain (no hydrolysis). B Feulgen/Gi- emsa's stain 77 ileria parasites were visible as small, densely staining, In the case of azure staining with hydrolysis, feeding sparse granules in a restricted area in an unstained back- of ticks for 1 or 2 days did not increase the definition of ground. The infected acini were not prominent but could the Theileria parasites, but there was a significant in- be recognized in conjunction with the presence of en- crease in the size of most of the infected acini that en- larged, violet-staining nuclei that differed from the nu- hanced the identification of infected cells. Similar results clei of uninfected acinar cells, which stained a mid-blue. were obtained with methylene blue staining with hydro- Azure staining with hydrolysis allowed the identification lysis. of Theileria parasites in unfed ticks as they stained In the case of MGP I staining, clear recognition of in- densely blue and were distinctly delineated with no other fected acini was not possible until ticks had fed for at structure being visible. Uninfected acini had small nuclei least 3 days because of a strong red to bluish-red staining in a pale blue to colorless cytoplasm. Methylene blue of uninfected acinar-cell cytoplasm. With MGP lI, after staining with hydrolysis produced results similar to those up to 2 days of tick feeding the Theileria parasite masses obtained using azure staining with hydrolysis; in con- appeared as indistinct, pale bluish-red areas that were trast, although methylene blue staining without hydroly- partially obscured by the reddish-stained cytoplasm of sis allowed the visualization of parasites, these were dif- the acinar cells. The enlarged, blue-staining nuclei could ficult to detect due to the heavy background staining of also be obscured by parasite masses. Beginning on day 3 cytoplasm in unfed ticks. of feeding, Theileria parasites became visible as blue- In conclusion, azure stain without hydrolysis allowed staining masses with a purple tinge that could obscure the clearest visualization of Theileria parasites in unfed the enlarged blue-staining nuclei of the infected acinar tick salivary glands, and the standard Feulgen's and cell. However, infected acini were easily recognizable. MGP stains were not useful for detection of parasites in Azure and methylene blue staining without hydrolysis the salivary glands of unfed ticks. did not increase the clarity of parasite staining.

Staining of salivary glands of ticks infected Staining of salivary glands of ticks infected with T. parva after feeding for 1-3 days with T. parva after feeding for 4 days

With Feulgen's stain, no distinct staining of Theileria With Feulgen's stain, Theileria parasites were distinct, parasite masses were seen until the ticks had fed for at staining dark red with either a slightly granular or homo- least 1 day. After that time the parasite masses appeared geneous appearance. Stained Theileria masses usually as reddish granules in an unstained or pinkish back- occupied more than half of the total acinar cell. Under ground. The nuclei of the infected cells were seen to be higher magnification, individual sporozoites could usual- enlarged and usually stained distinctly, which allowed ly be distinguished. The nuclei of infected acinar cells their additional differentiation from uninfected acinar were distinct and enlarged and stained dark red. Unin- cells. Uninfected acinar cells had red nuclei and a color- fected acinar-cell nuclei stained red, but the cytoplasm less or pale pink cytoplasm. After 2 days of feeding the was colorless or stained pink. In the case of combined density of the Theileria parasites, visible as red granules, Feulgen/Giemsa's stain, the staining of Theileria masses increased considerably. By the 3rd day of feeding the ap- was dark blue and distinctive (Fig. 2A). The stained The- pearance of Theileria masses was more homogeneous ileria masses usually covered most of the infected than granular and allowed clear identification of infected acinus, and the nuclei of the infected acinar cells were acini since the cytoplasm of uninfected acinar cells re- enlarged, distinct, and dark blue-staining and were not mained unstained or pink. usually obscured by the parasite masses (Fig. 2A). The With Giemsa's stain, after 1 day of feeding, the The- uninfected acinar-cell nuclei stained dark blue with a ileria parasites were visible as dark blue granules against pink- to red-colored cytoplasm. Giemsa's stain gave a re- a pale blue background. The nuclei of infected acinar sult similar to that obtained using Feul~en/G~emsaly " ' s cells were seen to be enlarged, stained violet-blue, and stain, as did toluidine blue staining. were clearly distinguishable from the nuclei of uninfect- Azure staining with hydrolysis showed the Theileria ed cells. By the 2nd day of feeding, Theileria masses masses as densely staining bodies that were blue to pur- stained intensely, with a dense granular or homogeneous ple and distinctly delineated. The nuclei of infected cells appearance allowing clearer diagnosis. FeulgerdGiemsa's were usually not visible. Uninfected acinar cells had stain produced a staining quality similar to that of Gi- small nuclei in a pale blue to colorless cytoplasm. Most emsa's alone but usually enabled a better distinction of of the infected acini were greatly enlarged, particularly infected acini in ticks fed for less than 2 days. when acetone and Carnoy's fixatives were used. The re- The density and intensity of the staining was more in- sults of methylene blue staining with hydrolysis were tensely violet as feeding of the tick proceeded with tolui- similar to the staining characteristics of azure (with hy- dine blue staining. By the 3rd day of feeding, Theileria drolysis). masses stained homogeneously and allowed a very clear With MGP I the staining of Theileria masses was dis- identification of parasites, as the cytoplasm of uninfected tinct, consisting of a blue granular to an almost homoge- acinar cells remained pale. neous appearance (Fig. 2B). Stained Theileria masses 78 Fig. 2A-D Staining of sali- vary-gland acini of R. append- iculatus infected with T. parva after 4 days of tick feeding (magnification, x150). The in- fected acinar cells are indicated with arrows. It can be seen that the salivary-gland acini become enlarged during feeding, in par- ticular the infected acinar cells. A Fenlgen/Giemsa's stain. B MGP stain I. C MGP stain II. D Azure stain (no hydrolysis)

normally occupied more than half of the hemisphere of inar cells had a homogeneous appearance, and their nu- the acinus. Nuclei of cells were seen to be enlarged but clei were enlarged and sometimes partially obscured by were usually obscured by parasite masses and were the parasite masses. Uninfected acini had blue nuclei and stained blue. Uninfected acini had blue-staining nuclei a red cytoplasm. and the cytoplasm stained a strong red that partially ob- Azure and methylene blue staining with hydrolysis scured the nuclei. MGP II stained Theileria masses dis- stained infected acini in a similar way. The Theileria par- tinctly blue with purple tinges (Fig. 2C). The infected ac- asite masses appeared as fairly densely stained, distinctly 79 delineated blue areas in which other structures were not tection of T. parva-infected acini as was Feulgen's stain visible (Fig. 2D). Uninfected acini had small nuclei in a on the salivary glands of ticks fed on rabbits for 4 days. pale blue to colorless cytoplasm. Most of the infected This finding should be confirmed using field ticks, as it acini were enlarged, which enhanced their identification. would make the assessment of field-tick batches much easier. For the reliable demonstration of infected acini it is Comparison of T. parva infection in two halves essential to use a method suitable for demonstrating all of the salivary glands treated with different stains stages of the parasite, regardless of whether the ticks have fed. Staining methods are required that help identi- When the salivary glands of infected male and female fy all stages of Theiteria reliably in the tick salivary ticks were separated into left and right halves and both glands. The ten staining methods investigated proved were stained in MGP I stain, there was no significant dif- suitable for the detection and quantitation of T. parva in ference between the infection in the right and left halves salivary-gland acini of R. appendiculatus that had fed for as determined using an ANOVA test (nested design). 4 days, and all were suited for use under laboratory con- However, there was a significant difference in the levels ditions. Several stains allowed satisfactory detection of of infection observed between the sexes, with females Theileria salivary gland stages in unfed ticks; in order of having more severe infections. When azure, MGP I, and efficiency, these were azure stain without hydrolysis, Feulgen's stains were compared for their ability to detect azure stain with hydrolysis, Feulgen/Giemsa's stain, Gi- infection in 4-day-fed ticks using the two halves of the emsa's stain, toluidine blue stain, and methylene blue salivary glands, there was no significant difference in stain without hydrolysis. It must be noted that the stan- levels of infection as determined using the Mann-Whit- dard laboratory stains, Feulgen's and MGR are not use- ney nonparametric test. ful for detecting parasites in unfed ticks. These stains, detecting sporoblasts in unfed ticks, are likely to be use- ful for assessment of the infection rate of ticks collected Discussion in the field either from vegetation or from animals, where the maturity of sporoblasts is unknown. The first Accurate quantitation of Theileria parva infection in the three stains could be used to quantitate infection accu- salivary glands of Rhipicephalus appendiculatus is of rately in unfed ticks. The azure stain without hydrolysis importance for experimental and epidemiology studies. can be recommended for use directly in the field when a In laboratory studies, this would allow the accurate rapid identification of infection is required or if there are choice of tick batches for experimental use. For example, difficulties in transporting the ticks to the laboratory be- to harvest the large numbers of Z parva sporozoites re- cause it is reliable, fast, and simple. Technical require- quired for biochemistry, molecular biology, and immu- ments for this method would be limited to a dissecting nology studies, which is of particular importance as the microscope, dissecting equipment, slides, and the simple circumsporozoite antigen, p67, is a candidate vaccine staining reagents. protein (Musoke et al. 1992), highly infected tick batches For the reason described in Results, MPG is not used have to be identified. Also, accurate assessment of low as a routine method in our laboratory, even for fed ticks. infection levels is required to choose tick batches resem- The morphology of the parasites is often not clear, and bling infection in field ticks for the challenge of immu- this stain is poorly suited for the detection of immature nized cattle. sporoblasts. T. parva-infected acini have been misidenti- The accurate assessment of T. parva infection in field fled by using this method. The method of Bascher (per- ticks is also important, as it would provide further insight sonal communication) appears to be more satisfactory, as into the epidemiology of T. parva infection and allow the parasite stages stand out more clearly. site-specific and general models of transmission to be Feulgen's method allows easy recognition of infected developed (Medley et al. 1993). At present there are ticks fed from day 2 onward, even for inexperienced per- some difficulties in assessing field infection that have sonnel. The simplified method of Btischer and Otim been discussed by Walker et al. (1981) and Young and (1986) and Bfischer and Tangus (1986) allows a more Newson (1973). Random collection of ticks from cattle rapid application than do the original methods of Blewett is not useful since the ticks have fed for unknown peri- and Branagan (1973) and Young and Leitch (1981). ods and many sporozoites may have been emitted. The Apart from being unreliable for the detection of T. parva use of bait cattle with the removal of ticks at daily inter- in ticks that had fed for less than 2 days, this stain has vals and their transfer to rabbits has been used, but the the additional disadvantage of requiring good-quality re- mortality of ticks can be high. A better method is to ob- agents, particularly Schiff's reagent, which are often not tain ticks from vegetation that are obviously questing for available in simple tropical laboratories. All the stains hosts. A significant claim in the present study is that require reagents that may have to be obtained for the azure stain can be used to detect infected salivary-gland specific purpose of infection-rate studies in ticks, with acini accurately in unfed ticks. It has been shown in ex- the exception being the modified Giemsa's stain, for perimentally infected ticks that azure stain applied to the which only very basic components are required and are salivary glands of unfed ticks was as accurate in the de- usually available in laboratories in the tropics. This 80 method also allows the easy recognition of all salivary- Acknowledgements Thanks are due the staff of the ILRAD tick gland stages of Theileria, making it useful for field stud- laboratory for expert technical assistance in dissection and stain- ing (Messrs. B. Otim, W. Ratemo, and J. arap Tangus) and animal ies. handling (Messrs. K. Kariuki, P. K. Mburu, and P. Macharia). This Feulgen's-type hydrolysis in a hot solution of mineral report is ILRAD paper 1280. acid followed by staining with basic dyes is used for the specific metachromatic staining of certain types of endo- crine tissues, such as argyrophils or G-cells of the References pyloric antrum (Pearse 1985). Attempts were made to simplify the procedure for salivary gland staining by us- Bailey KP (1960) Notes on the rearing of Rhipicephalus append- ing hydrolysis at room temperature. The complete range iculatus and their infection with Theileria parva for experi- of basic dyes have not been fully explored and there is a mental transmission. Bull Epizoot Dis Afr 8:33-43 scope for further investigation of dyes, such as using Bishop R, Sohanpal B, Kariuki DE Young AS, Nene V, Baylis H, metachromatic fluorescent basic dyes under the fluores- Allsopp BA, Spooner PR, Dolan TT, Morzaria SP (1992) De- tection of a carrier state in Theileria parva infected cattle us- cent microscope as a means of not only detecting but ing polymerase chain reaction. Parasitology 102:347-353 possibly identifying species of Theileria and Babesia in Bishop RP, Sohanpal B, Morzaria SP, Dolan TT, Mwakima FN, the salivary glands of their vectors. The mode of action Young AS (1994) Discrimination between Theileria parva and of basic dyes after mineral acid hydrolysis has been dis- T. taurotragi in the salivary glands of Rhipicephalus append- cussed by Pearse (1985). After Feulgen's staining, reac- iculatus ticks using oligonucleotides homologous to ribosomal RNA sequences. Parasitol Res 80:259-261 tive components remain available for further reaction Blewett DA, Branagan D (1973) The demonstration of Theileria with blue-staining components in Giemsa's stain as parva infection in intact Rhipicephalus appendiculatus sali- shown for the Feulgen/Giemsa's method described here- vary glands. Trop Anim Health Prod 5:27-34 in. This is an important step in this method because poor Brocklesby DW, Barnett SF, Scott GR (1961) Morbidity and mor- tality rates of East Coast fever (Theileria parva infection) and uptake of stain due to a poor quality Schiff's reagent can their application to drug screening procedures. Br Vet J lead to some difficulty in the identification of infected 117:529-531 acini. In this case, instead of repeating the whole proce- B%cher G, Otim B (1986) Quantitative studies on Theileria parva dure, counterstaining with Giemsa's stain on Schiff's im- in the salivary glands of Rhipicephalus appendiculatus adults: quantitation and prediction of infection. Int J Parasitol proves the quality of the first stain such that the positive 16:93-100 acini become identifiable. Bfischer G, Tangus J (1986) Quantitative studies on Theileria par- The superior reaction of azure with infected salivary- va in the salivary glands of Rhipicephalus appendiculatus gland tissues from unfed ticks in the absence of hydroly- adults: search for conditions for high infections. Int J Parasitol sis could be partially explained by the extreme acidity of 16:121-129 Chen PR Conrad PA, Ole-Moiyoi OK, Brown WC, Dolan TT the solutions used. The pH ranged from 2.6 for the con- (1990) DNA probes detect Theileria parva in the salivary centrated solution (1%) to 4.4 for the 0.1% dilution used gland of Rhipicephalus appendiculatus ticks, Parasitol Res in these studies. It was also possible to use this stain suc- 77:590-594 cessfully without any fixation prior to staining. Cowdry EV, Ham AW (1932) Studies on East Coast fever. The life cycle of the parasite in the tick. Parasitology 2:1-49 One problem that can be encountered is that in the Dolan TT, Young AS, Losos GJ, McMillan I, Minder CE, Soulsby field in eastern, central, and southern Africa, R. append- K (1984) Dose dependent responses of cattle to Theileria par- iculatus may be infected with at least two species of The- va stabilate infection. Int J Parasitol 14:89-95 ileria: T. parva and T. taurotragi. New technologies us- Fawcett DW, Doxsey S, Bfischer G (1982) Salivary gland of the tick vector of East Coast fever, lII. Ultrastructure of sporogony ing either DNA or RNA probes on squash preparations in Theileria parva. Tissue Cell 14:183-206 of dissected salivary glands have proved to be useful in Fawcett DW, Young AS, Leitch BL (1985) Sporogony in Theileria the detection of T. parva in salivary glands (Chen et al. (: ). A comparative ultrastructural 1990; Bishop et al. 1994) and for the differentiation of T. study. J Submicrosc Cytol 17:299-314 parva and T. taurotragi in the salivary glands (Bishop et Goddeeris BH, Katende JM, Irvin AD, Chumo RSC (1982) Indi- rect fluorescent antibody test for experimental and epidemio- al. 1992). The sensitivity of these probes needs to be es- logical studies on East Coast fever (Theileria parva infection tablished, but they may not be useful for quantitation of in cattle): evaluation of a cell culture schizont antigen fixed the abundance of infection in field ticks. In addition, the and stored in suspension. Res Vet Sci 33:360-365 polymerase chain reaction using Theileria species-spe- Irvin AD, Boater CDH, Dobbelaere DAE, Mahan SM, Masake R, Ocama JGR (1981) Monitoring Theileria parva infection in cific oligonucleotide primers has been shown to detect adult Rhipicephalus appendiculatus ticks. Parasitology infection soon after the salivary glands have been pene- 82:137-147 trated by Theileria kinetes and can be used to differenti- Kok JB de, D'Oliveira C, Jongejan F (1993) Detection of the pro- ate species (Bishop et al. 1992; de Kok et al. 1993). tozoan parasite Theileria annulata in Hyalomma ticks by the polymerase chain reaction. Exp Appl Acarol 17:839-846 DNA probes are used with radioactive labelling at Lessard P, L'Eplattenier R, Norval RAI, Kundert K, Dolan TT, present but would become practical in laboratories with Croze H, Walker JR Irvin AD, Perry BD (1990) Geographi- limited facilities if nonradioactive labels could be ap- cal information systems for studying the epidemiology of plied to these probes. It is expected that the improved cattle diseases caused by TheiIeria parva. Vet Rec staining method for the determination of infection levels 126:255-262 Martin HM, Barnett SF, Vidler BO (1964) Cyclic development in ticks will complement the newly developed nuclei ac- and longevity of Theileria parva in the tick Rhipicephalus ap- id-based detection systems. pendiculatus. Exp Parasitol 15:527-555 81 Medley GF, Perry BD, Young AS (1993) Preliminary analysis of Walker AR, Young AS, Leitch BL (1981) Assessment of Theileria transmission dynamics of Theileria parva in eastern Africa. infections in Rhipicephalus appendiculatus ticks collected in Parasitology 106:251-264 the field. Z Parasitenkd 65:63-69 Musoke A, Morzaria S, Nkonge C, Jones E, Nene V (1992) A re- Young AS, Leitch BL (1981) Epidemiology of East Coast fever: combinant sporozoite surface antigen of Theileria parva induc- some effects of temperature on the development of Theileria es protection in cattle. Proc Natl Acad Sci USA 89:514-518 parva in the tick vector Rhipicephalus appendiculatus. Para- Pearse AGE (1985) Histochemistry: theoretical and applied, vol II, sitology 83:199-211 4th edn. Churchill Livingstone, Edinburgh Young AS, Newson RM (1973) An improved method of handling Purnell RE, Joyner LP (1968) The development of Theileria parva ticks collected in the field. Res Vet Sci 15:133-135 in the salivary glands of the tick Rhipicephalus append- Young AS, Grootenhuis JG, Leitch BL, Schein E (1980) The de- iculatus. Parasitology 58:725-732 velopment of Theileria=Cytauxzoon taurotragi (Martin and Steel RGD, Tonie JH (1980) Principles and procedures of statis- Brocklesby I960) from eland in its tick vector RhipicephaIus tics. A biometrical approach, 2nd edn. McGraw-Hill, New York appendiculatus. Parasitology 81:129-144 Walker AR, McKellar SB, Bell LJ, Brown CGD (1979) Rapid Young AS, Leitch BL, Stagg DA, Dolan TT (1983) Identification quantitative assessment of Theileria infection in ticks. Trop of Theileria infections in living salivary glands of ticks. Para- Anita Health Prod 11:21-26 sitology 86:519-528