A Rapid Method to Select Rhipicephalus appendiculatus Salivary Glands Infected with Theileria parva

Abstract

Light microscopic studies on the detection and development of the salivary gland stages of Theileria parva in feeding adult ticks, Rhipicephalus appendiculatus, have involved the use of various stains such as Feulgen (Blewett and Branagan, 1973, Trop. Anim. Hlth Prod. 5:27-34), Giemsa (Pumell and Joyner, 1968, Parasitology 58: 725732) and Methyl Green Pyronin (MGP) (Walker et al., 1979, Trop. Anim. Hlth Prod. 11: 21-26; Irvin et al., 1981, Parasitology 82: 137-147). Although the method developed by Irvin et al. (1981, loc. cit.) significantly increased the speed with which parasites could be detected and examined, it still involved the dissection and teasing out of glands onto a microscope slide, followed by fixing, rinsing, staining, mounting in DPX under cover-slips and examination by a light microscope. We report here the development of a method which allows detection and selection of heavily infected salivary glands based on direct staining of a small part of 1 salivary gland in a drop of MGP. When adult ticks were immobilized in paraffin wax and the cuticle and gut removed, it was possible to fix and stain the salivary glands without removing them from the tick and thus detect T. parva-infected salivary gland acini using a dissection microscope at x 50 magnification. Carnoy's fixative (absolute ethanol 60%, chloroform 30%, glacial acetic acid 10%), was placed on each dissected tick for 5 min, followed by a rinse with 70% ethanol and one rinse with distilled water for 2 min each. Staining was carried out with a drop of MGP (aqueous Methyl Green 0.04%, aqueous Pyronin Y 0.02% in 0.5 M acetate buffer, pH 4.8) for 8 min. Nuclei of uninfected acinar cells stained blue whereas their cytoplasm containing RNA stained pink. The cytoplasm of infected cells containing parasite nuclear chromatin stained blue, allowing them to be distinguished from uninfected cells. In attempts to further simplify the method, it became clear that fixation prior to staining was not necessary for detection of the parasite. This observation then led to the development of a protocol for the selection of heavily infected salivary glands. Salivary glands infected with T. parva were obtained by feeding R. appendiculatus nymphs on cattle reacting to stabilate-induced infection (Cunningham et al., 1973, Int. J. Parasit. 3: 583587). Four-day fed adult female and male ticks were mounted in paraffin wax in a Petri dish and individual ticks numbered in the wax near the mouthparts. As many as 100 ticks could be mounted in 1 Petri dish. The dissection medium usually consisted of Dulbecco's phosphate buffered saline pH 7.4 (PBS), but other media such as Hanks' Balanced Salt Solution, Dulbecco's Minimum Essential Medium or Leibovitz's L1 5 could also be used. The dorsum and gut of the ticks were removed and the dissecting medium replaced by fresh PBS. A small piece of salivary gland was removed from every tick and deposited in a drop of PBS in a numbered well in the top row of a Wellcome PTFE Multispot slide (C. A. Hendley, Essex, UK), the numbers of the wells corresponding with the numbers of the ticks. The wells in the bottom row of the slide contained a drop of MGP. All salivary gland pieces were then transferred to the wells in the bottom row containing the MGP and left to stain for 8 min before transfer back to the original drops of PBS on the top row of the slide. Glands were examined and their infection level assessed. Salivary gland pieces from 15 to 20 ticks were routinely processed at a time. The numbers of the glands that contained many infected acini were recorded. When screening of ticks was completed and the numbers of all heavily infected ticks recorded, the remainder of the gland from which a sample was taken, together with the second gland, were removed for experimental use. Particular advantages of this method are its rapid process and minimal risk of losing or damaging the infected glands. This technique has been successfully applied for the selection of heavily infected tick salivary glands used for immunizing mice for the production of anti-sporozoite mon-