Erperimental & 4pp/ied Acarology, 17 ( 1993 ) 839-846 839

Detection of the protozoan parasite Theileria annulata in Hyalomma ticks by the polymerase

chain reaction

J.B. de Kok, C. d'Oliveira and E Jongejan* Tick Research Unit. Department (![Parasitology and Tropical Veterinaty Medicine. Faculty of

Veterinary ,14edicine, Umver~'io" ~!/ Utrecht, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands

(Accepted 1 December 1993)

ABSTRACT

de Kok, J.B., d'Oliveira. C. and Jongcjan, F.. 1993. Detection of the protozoan parasite Theileria atmulata in Hyalomma licks by the polymcrase chain rcactmn. Exp. Appl. Acarol.. 17: 839-846.

Adult Hvalomma ticks were examined l~)r the presence of Theileria amudata infection using the Polymerase Chain Reaction IPCR). A 372 bp DNA fiagment derived from lhe small ribosomal RNA gene cff 7" anmdata was anaplitied from 45 out of 50 (90%) H. dromedarii ticks and from 36 out of 50 (72%) H. mal3matum marginatum ticks. No product was amplilied from non-infected control ticks. Restriction enzyme digestion with Sac H confirmed that the product was derived from the largeted 7: amudata gene. As a further confirmation it was shown daat ba~th species of Ityalomma ticks were able to Iransmit 7'. amndata to experimental calves. PCR detection of TheileHa parasites in ticks was compared with conventional staining of dissected salivar3, glands using methyl green pyronin and its comparative advantages are discussed.

INTRODUCTION

Theileria annulata is a tick-borne protozoan parasite which canscs tropical theileriosis in cattle. The disease is widcly distributed ranging from southern Europe and northern Africa, through the Middle East into central Asia and is characterized by a lympho-prolifcrative and lympho-destructivc phase, usually accompanied by a marked anacmia (Uilenbcrg, 1981). T. annulata is transmitted by ticks of the genus Hvalomma (Robinson, 1982) and an estimated 250 million domestic cattle are at risk fiom the disease (Tait and Hall, 1990). The parasite enters the bovine host during tick feeding as sporozoites, which rapidly invade lymphocytes, where they maturc into macroschizonts and induce proliferation of the host cell. Develop- ment of macroschizonts into microschizonts results in the relcasc of merozoites. which invadc erythrocytes and subsequently mature into piroplasms.

"To whom conespondence should be addressed.

© Science and Technology Letters. All rights reserved.

8 4 0 J.a. DE KOK FT" AI..

Several methods have been developed to determine the infection rates of T. annulata sporozoitcs within vector tick populations. For instance, Theileria sporo- zoites can be detected within salivary gland acini of infected ticks by methyl green pyronin (MGP) staining (Walker et al., 1979; 1983). Although this method is rela- tively easy to perform, it is not possible to differentiate between sporozoites belong- ing to different Theileria species. This is important, for instance for mixed infections of T. annulata and T. lestoquardi sporozoites in Hyalomma ticks (Morel and Uilcn- berg, 1981 ). Moreover, MGP cannot be used on alcohol preserved tick specimens.

The polymerase chain reaction (PCR), which uses specific oligonucleotide primers and Taq DNA polymerase to synthesize a large number of copies from a single DNA template (Saiki et al., 1988), has very rapidly become a standard lab- oratory technique. PCR has shown to be a valuable tool in the detection and identi- fication of micro-organism within infectcd tick vectors, such as Borrelia burg- dorferi spirochetes within lxodus ticks (Persing et al., 1990; Johnson et al., 1992).

Recently, the ccntral region of the small subunit ribosomal RNA (srRNA) sub- unit of different Theileria species was sequenced (Gajadhar et al., 1991; Ails0pp et al., 1993), providing specific primers for PCR amplification of T. annulata DNA (AIIsopp et al., 1993). In this paper, we used thesc oligonuclcotide primcrs for the amplification of T. annulala DNA within infected Hyalomma ticks and compared the sensitivity of PCR with MGP staining of salivary glands. The ability of the ticks to transmit T. annulata was proven by transmission of tropical theileriosis to susceptible calves.

M A T E R I A L S A N D M E T H O D S

77ck colonies A laboratory colony of non-infected Hyalomma dromedarii ticks was established from engorged females collected by M. van Stratcn from camels at Arava, Israel (Van Straten and Jongcjan, 1993). 7: annulata infected adult H. marginatum marginatum ticks were received from Dr. M. Habela, University of Extremadura, Caceres, Spain. These ticks transmitted T. annulata (Caceres isolate) to two Fricsian calves (nos. 287 and 226). For acquisition of the infection, larvae of H. m. marginatum were placed on calf no. 226 and those of H. dromedarii on calf no. 287. Following drop off, the engorged nymphs were allowed to moult at 27°C with approximately 85% relative humidity. Post moulting development was at 18°C and 85% humidity. H. dromedarii wcre stored for 6 months and H. m. marginatum for 3 months at 18°C before they were used for methyl green pyronin staining or stored for at least 24 hours in 70% ethanol for subsequent PCR analysis. Only alcohol- fixed specimens were cxamincd.

T. annulata in salivar3. ~ glands Prior to MGP staining, stored adult H. dromedarii ticks were incubated for 4 days at 37°C and 100% humidity to induce sporozoite maturation (Samish, 1977).

DETECTION OF THE PROTOZOAN PARASITE THEILERL4 A:VNUl_417~ IN HYALOM,iL,I TICKS 841

Saliva~, glands were dissected and gently teased apart on a microscope slide in a drop of bovine serum albumen ( 1% w/v). Slides were air-dried and stained by accustain methyl green pyronin solution (Sigma Diagnostics, St. Louis. USA) according to Walker et at. (1979).

Preparation qf ticks for PCR analysis Ticks were removed from 70% ethanol and air-dried on filterpaper for 5 min and were cut in half using a sterile blade. For each tick a new blade, a heat stcrilizcd forceps and a new piece of filterpaper were used in order to prevent cross con- tamination. Both halves of each tick were put in the same screw-top vial (Multi Tcchnology Inc., Salt Lake City, UT, USA) containing 0.5 mm and 0.1 mm diameter zirconium beads (approx. 200 txL of each) (Biospec Products, Bartles- ville, OK, USA) and 300 txL buffer I (10 mM TRIS-HC1 (pH 8.3), 50 mM KC1, 2.5 mM MgC12). Tick tissue was crushed tbr 190 seconds using a Mini-Beadbeater (Biospcc Products, Bartlesville, OK, USA) (Stouthamer et al.. 1993). Subsequently, 100 ~L of the supernatant was centrifuged for 2 min at 13,000 rpm. The pellet was resuspended in 100 p~L lysis-buffer (buffer 1 supplemented with 0.01% gelatin, 0.45% NP40 and 0.45% Tween 20). To this mixture 12 txg proteinase K was added, mixed, incubated for 60 rain at 56°C, inactivated for 10 rain at 95°C and im- mediately put on ice. After mixing, the suspension was centrifuged for 1 min at 13,000 rpm and the supernatant was either stored at -20°C or immediately, used for PCR.

Polymerase chain reaction A pair of oligonucleotide primers (primer 1 (Theileria-specific): 5' AGT-FTCT- GACCTATCAG 3' and primer 2 (Theileria annulata-specific): 5' TGCACAGAC- CCCAGAGG 3') was synthesized, deduced from the small ribosomal RNA gene of T. annulata (Allsopp et al., 1993). The length of the thcilerial genome t~u:geted lk)r amplification was predicted to be 372 bp. PCR reactions were performed in a 0.5 mL GeneAmp TM reaction tube (Per'kin-Elmer Cetus, Norwalk, CT, USA) con- taining the following mixture: 120 ng of each primer, 200 gM of each nucleotide triphosphate, 4.0 mM MgCI 2, 1 p.L of template, and 0.5 units of Taq polymerase (Promcga, WI, USA) in 10 mM TRIS-HC1 (pH 8.0). 50 mM KCI. Sterile double distilled water was added to a final volume of 100 gL. Alter mixing, 100 gL of sterile paraffin oil was added to avoid evaporation. PCR was performed in a Perkin Elmer Cetus DNA Thermal Cycler and each run consisted of 40 cycles using the following parameters: denaturation at 94°C (1 rain), annealing at 60°C (1 rain) and extension at 72°C (I rain).

Detection and identi[ication of the PCR product Ten lxL of each reaction mixture were anatyscd by get clectrophoresis using a 1.6% agarose gel which was stained by ethidium bromide, according to standard methods (Maniatis et al., 1989). Genomic DNA purified from piroplasms of the

842 J.B. DI- KOK ET AL.

Ankara strain of T. annulata was used as positive control. Specificity of the 372 bp amplificd DNA fragment was verified by restriction enzyme digestion using Sac H (Pharmacia, Uppsala, Swcden) and Southcrn blot hybridization with a Theileria- specitic 18-met internal probe (5' GGAAGGCAGCAGGCGCGC 3'), which was labelled with digoxygenine using a DIG Oligonucleotide Y-End Labeling Kit (Boehringer Mannheim, Mannheim Gmbh, Germany). Briefly, the DNA-fragmcnts were transferred onto Nytran TM (Schleicher & Schuell, Germany) and hybridized with the labclled intcrnal probe at 54°C in 5x SSC (0.8 M NaCI, 0.08 M Na- citrate, pH 7,0), 0.1% N-lauroylsarkosine, 0,02% Sodium-Dodccyl-Sulphatc. The DNA fragment was detected with a DIG Luminescent Detection Kit (Bochringer Mannheim).

Tick transmission ofTheilc'ia annulata to experimental calves Fifty adult H. dromedarii ticks (25 males and 25 females) from thc same batch as used for PCR and MGP staining were applied in an earbag of Friesian calf no. 292 and 50 adult H. m. marginatum ticks were applied onto Friesian calf no. 293. The infections were monitored by daily temperature records, clinical observations and microscopic examination of macrosehizonts and theilerial piroplasms in Giemsa stained lymphnode biopsies and blood smears.

RESULTS

Fifty infected adult H. dromedarii ticks and 50 infected adult H. m. marginatum ticks were examined by PCR. Thirty non-infected H. dromedarii ticks were used as negative controls h)r both tick batches. An cxpccted 372 bp fragment of the srRNA gene of T. annulata was amplified in 45 out of 50 (90%) H. dromedarii ticks and in 36 out of 50 (72%) H. m. marginatum ticks (Table 1, Figs 1 and 2). No product was ~unplified from thirty non-infected H. dromedarii ticks.

Restriction enzyme digestion (Fig. 3) and Southern blot analysis (not shown) confirmed that the product was derived from the tmgeted T. annulata genc. Using

"I'ABI,E 1

Theiteria annulata detection in Hyalomma ticks: methyl green pyronin staining (MGP) versus PCR.

Non-int¢cted In footed

llyalomma dromedarii Hyalomma dromedarii Hyalomma m. marginamm

MGP PCR MGP PCR MGP IRTR Positive 0 0 32 45 nd* 36 Negative I 0 30 5 5 nd* 14 Percentage 0 0 86 90 nd* 72

* = not done

DETECIION OF IIIE PROTOZOAN PARASITE TI-IEILERL.t A.\:NUIA1?I IN IIYALOMMA TICKS 843

Fig. 1. Detection of T, ammlata in infected H. dromedarii ticks by PCR. I,ane m. molecular size marker*. Lane 1, no DNA template (negative control), Lane 2, non-infected tick sample, Lanes 3 and 5 to 14. individual infected tick samples. Lane 4, purified piroplasm DNA (positive control), *tth~fl digested pBluescript size standard (in bp); 1077, 517,456, 396, 356, 75.

Fig. 2. Detection of 7: annulata in infected H. m. marginatum ticks by PCR. Lane m, molecular size marker*, L,'me 1, no template. Lane 2. non-infected H. dromedarii tick sample, Lane 3, purified piro- plasm DNA (positive control). Lanes 4 to 12, individual infected tick samples. *Hi~/ l digested pBlue- script size standards (in bp): 1077, 517,456, 396, 356. 75.

restriction enzyme Sac H. which cuts at an unique site, two fi'agments of 273 and 100 bp were obtained as expected (Fig. 3). Southern blot analysis was performed on negative and positive PCR results derived from 39 H. m. marginatum and 4 H. dromedarii ticks. Ticks that were negative on gel electrophoresis remained negative on Southern blots, whereas positive PCR results could be confirmed by Southern analysis, Thirty-two out of 37 (86%) of the ft. dromedarii ticks were

~44 J.B. DE KOK VI" AI..

infected with Theileria using MGP staining (Table 1). The parasite masses varied greatly between individual ticks. H. m. marginatum ticks were not examined by MGP staining.

Both H. dromedarii ticks on calf no. 292 and H. m, marginatum tick on calf no. 293 were able to transmit T. annulata. Calf no. 293 required treatment with Butalex (Coopers Animal Health Ltd, Bcrkhamsted, England), while the other calf recovered spontaneously. Further parasitological parameters are given in Table 2.

Fig. 3. Sac H restriction enzyme digestion of the PCR amplified 372 bp fragment of the 7~ annulata specilic srRNA gene. Lane m, molecular size marker*. Lanes I, 3, 5, 7, undigested fragments, lmnes 2, 4, 6, 8, corresponding digested fragments. Samples shown in Lanes 1 and 3 of Fig. 3 correspond to those shown in Lanes 9 and 10 of Fig. 1, respectively. Samples shown in Lanes 5 and 7 of Fig. 3 correspond to those of Lanes 4 and 7 of Fig. 2, respectively. * H i n f l digested pBluescript digested size standards (in pb); 1077, 517, 456, 396, 356, 75.

TABLE 2

Transmission of 177eileria annulata to experimental calves by 14yalomma ticks.

Calf Tick No. of ticks No. of ticks Days to Days to Days to no. species applied engorged temperature microschizonts piroplasms

292 tl. dromedarii 25 ¢3 22 9 7 10 13 * 25

293 tI. m. marginatum 25~ 189 10 11 13 25 ?

*Calf treated with Butalex on day 14.

DISCUSSION

T. annulata was successfully amplified from Hyalomma ticks using the Polymerase Chain Reaction. The method was tested on two known experimental vector species of T. annulata. Although H. dromedarii had not been considered to play an im- portant role in the transmission of T. annulata in the field, Um el Hassan Mustafa

DEIECIION OF TIlE PROTOZOAN PARASIIE THEII.ERbl AAWUL..t E4 IN HI'?ILOMMA TICKS 8 4 5

et al. (1983), this tick has recently been incriminated as a field vector of tropical theileriosis in Mauretania, where H. anatoticum anatolicum does not occur (Jacquiet et al., 1990). H. m. marginatum is considered to be the main vector of T. annulata in Spain (Habela et al., 1993). The method described here may also be applied to H. anatolicum anatolicum, which is considered to be the main vector of tropical theileriosis in the field.

Both resu'iction enzyme digestion and Southern blot hybridization confirmed that the PCR specifically amplified the expected 372-bp theilerial srRNA target. Neither amplification nor hybridization was observed in non-infected control ticks. Likewise, ticks that proved negative in gel-electrophoresis remained negative on Southern blot analysis. The variation in intensity between fragments amplified from tick samples in lane 6, 7 and 8 of Fig. 1 could be due to observed variations in para- site masses betwcen individual ticks as detected by MGP staining. The presence of T. annulata in both tick batches was further confirmed by the successful trans- mission of T. annulata to susceptible Friesian calves (Table 2).

Application of PCR to detect Theileria infection in vector ticks has several advantages over MGP staining of salivary glands. Unlike MGP staining, PCR does not require fresh specimens, but can be performed on alcohol preserved specimens as has also been demonstrated earlier for the detection of Borrelia in lxodes dammini ticks (Persing et al., 1990). Such versatility of PCR makes it very ad- vantageous in field studies by reducing the risks involved in the maintenance and transportation of infected live ticks. Moreover, PCR has shown to be useful to dis- tinguish between different species of Theileria (Alisopp et at., 1993), which is important in case mixed infections occur This is especially relevant for T. parva and T. taurotragi, which are both transnfitted by Rhipicephalus appendiculatus ticks in Eastern and Southern Africa. T. pan,a causes severe illness in cattle, whereas T. murotragi is relatively mild. Moreover, both parasites are not easily distinguished by serology and it has been very difficult to assess their relative importance in the field, for instance in Zimbabwe (Grootenhuis et at., 1979; Koch et al., 1988).

Although the sensitivity of MGP-staining (32 out of 37 positive (86%)) and PCR (45 out of 50 positive (90%)) was within the same range (Table 1), a more detailed comparison between both methods using more tick specimens is required. Finally, PCR detection of Theileria parasites in ticks has potential to become an important biotechnological tool in the epidemiology of theileriosis.

ACKNOWLEDGEMENTS

This research was supportcd by the European Community (Directorate General XII) STD-3 programme under contract no. TS3* - CT91-0019 entitled 'The Characterization of Protective Antigens and their genes from Theiteria annulata."

We are very grateful ~o Dr. B.A. Allsopp for access to unpublished work and to Dr. Miguel Habela for providing H. margmatum ticks infected with the Caceres isolate of T. annulata.

846 J,B. DE KOK El' AL.

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