Theileria Parva in the Salivary Glands of Rhipicephalu$ Appendiculatu, Evaluation of Staining Methods

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 Theileria 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 tick 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 ticks 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 infection 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 salivary 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 described above and additional staining with 10% Giemsa's stain solution (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 solution (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 maintained 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.

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