Veterinary Parasitology 113 (2003) 115–121

A comparison of susceptibilities to infection of fourspecies ofHyalomma ticks with Theileria annulata

F. Sayina,∗, Z. Karaera, S. Dincera, A. Cakmaka,A. Inci a, B.A. Yukaria, H. Erenb, Z. Vatansevera,

S. Nalbantoglua, T.R. Melroseca Department of Protozoology and Medical Entomology, Faculty of Veterinary Medicine,

University of Ankara, 06110 Ankara, Turkeyb Department of Parasitology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey

c Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies,Easter Bush, Roslin, Midlothian EH25 9RG, UK

Received 16 April 2002; received in revised form 3 January 2003; accepted 16 January 2003

Abstract

In this comparative study unfed nymphs of fourHyalomma tick species (Hyalomma anatolicumanatolicum, Hyalomma anatolicum excavatum, Hyalomma detritum andHyalomma marginatummarginatum) were allowed to engorge on calves experimentally infected withTheileria annulata.The infection prevalence in the salivary glands of the adult female and male ticks of eachHyalommaspecies used in the study were assessed. The infection prevalence withT. annulata was high anddid not vary markedly in the fourHyalomma tick species. The mean number of infected acini pertick in female and male ticks was different with female ticks having higher numbers of infectedacini than the male ticks. The sex difference was more significant betweenH.a. anatolicum andH.a. excavatum than betweenH. detritum andH.m. marginatum. This study clarifies the roles offour Hyalomma tick species, and their sex, in the development ofT. annulata.© 2003 Elsevier Science B.V. All rights reserved.

Keywords: Hyalomma anatolicum anatolicum; Hyalomma anatolicum excavatum; Hyalomma detritum;Hyalomma marginatum marginatum; Theileria annulata; Prevalence

1. Introduction

The protozoan parasiteTheileria annulata Dschunkowsky and Luhs, 1904 is transmittedfrom cattle to cattle by ticks of the genusHyalomma. SeveralHyalomma species have

∗ Corresponding author. Tel.:+90-312-317-0315; fax:+90-312-316-4472.E-mail address: parasite@veterinary.ankara.edu.tr (F. Sayin).

0304-4017/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0304-4017(03)00045-1

116 F. Sayin et al. / Veterinary Parasitology 113 (2003) 115–121

been implicated in the transmission of this parasite or are thought to be associated withdisease outbreaks in the field. The principal vectors ofT. annulata areHyalomma detritumSchulze, 1919 (Sergent et al., 1931; Gill et al., 1974: Samish and Pipano, 1976, 1978)andHyalomma anatolicum anatolicum Koch, 1844 (Dhar et al., 1982; Bhattacharyulu etal., 1975a,b; Walker and McKellar, 1983; Sangwan et al., 1989). It has been reported thatHyalomma anatolicum excavatum Koch, 1844 (Daubney and Sami Said, 1951; Samish andPipano, 1983), Hyalomma dromedarii Koch, 1844 (Mazlum, 1969; Bhattacharyulu et al.,1975a) andHyalomma marginatum marginatum Koch, 1844 (Bhattacharyulu et al., 1975a)also transmitT. annulata. Quantitative assessment ofT. annulata infection in the salivaryglands ofH.a. anatolicum has been made by several workers (Dhar et al., 1982; Reidand Bell, 1984; Walker et al., 1983, 1985) and considerable variation in both prevalence(the number of infected ticks in relation to the number of ticks in the population at riskof developing the infection) and intensity (abundance of sporoblasts in salivary glandsof infected ticks) ofT. annulata infection in salivary glands has been demonstrated. Theintensity and prevalence ofT. annulata infection in otherHyalomma species has not yetbeen investigated. For accurate studies on bovine tropical theileriosis it is necessary toassess the infection prevalence ofT. annulata in the endemic vector ticks as they may exerta considerable effect on the disease status of the host. In the present study a comparativeassessment has been made of both the infection prevalence and intensity ofT. annulatain the salivary glands of fourHyalomma species. The four speciesH.a. anatolicum, H.a.excavatum, H. detritumandH.m. marginatum are widespread in Turkey. As nymphs theywere engorged on calves experimentally infected withT. annulata and prevalence andintensity of the parasite infection in salivary glands of their adult females and males, wereassessed.

2. Materials and methods

2.1. Experimental calves

Holstein calves, approximately 3-month old and reared without exposure to ticks, wereused. Prior to inoculation withT. annulata by stabilated sporozoites, the examination ofblood smears and indirect fluorescent antibody test (IFAT) confirmed that the calves werefree from infection. After infection the rectal temperature of the calves was recordedand blood smears were examined daily. Lymph node smears were prepared by needlebiopsy when the nodes became enlarged. Blood and biopsy smears were stained withGiemsa stain and examined microscopically for the presence ofT. annulata, piroplasms orschizonts.

2.2. The parasites

Two blood samples infected withT. annulata were drawn from clinical cases of tropi-cal theileriosis, one in the village of Sarioba, about 100 km from Ankara, and the other inAkdere, a suburb of Ankara. Two susceptible calves were inoculated subcutaneously withthe infected blood samples either from Sarioba or from Akdere. UninfectedH.a. anatolicum

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nymphs were fed on the two calves after development of the infection. The infection preva-lence of the adult ticks infected with eitherT. annulata (Sarioba) orT. annulata (Akdere)was 100%. Ground up tick supernatants (GUTS) were prepared from both isolates (Brown,1987) and the infectivity of both GUTS was confirmed in susceptible calves prior to cry-opreservation in the gas phase of liquid nitrogen.

2.3. Infection of experimental calves

After removal from liquid nitrogen, the two tick stabilates ofT. annulata (Sarioba) andT. annulata (Akdere) were thawed in a water bath at 37◦C and allowed to equilibrateat room temperature for 30 min. One ml of the stabilate containing four tick equivalents(4 te/ml) was inoculated, subcutaneously, above the right prescapular lymph node of thecalves.

2.4. Ticks

Unfed nymphs of the fourHyalomma species were produced from the infection-freetick colonies (Walker et al., 1985) which have been maintained in Ankara University since1987.

2.5. Experimental infection of ticks

Unfed nymphs of all fourHyalomma species were fed separately on the ears or on thebacks of calves experimentally infected withT. annulata (Sarioba) orT. annulata (Akdere).In one experiment nymphs were fed on a calf infected with bothT. annulata (Akdere) andT. annulata (Sarioba) designatedT. annulata (Ak-Sa). The engorged nymphs were placedimmediately into a desicator maintained at 85% (PH) in an incubator at 28◦C for moulting(Walker et al., 1985). The unfed adult ticks remained in the same incubator for about 1week to get activity under the same conditions, then they were fed on a rabbit for 3 daysand dissected.

2.6. Examination and staining of tick salivary glands

Parasite development in the salivary glands of the moulted adult ticks was assessed after 3days feeding on rabbits. Ticks were dissected in 0.85% saline using a binocular stereo micro-scope. Both salivary glands were removed and stained whole by the Methylgreen/Pyroninmethod ofWalker et al. (1979). The stained salivary glands were mounted in DePeX andexamined using 40× magnification. The total number of acini containing parasite masseswas counted.

2.7. Statistics

The statistical test applied was the Mann–Whitney test,T-test andχ2-test to compare thetick species, female and male tick samples.

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3. Results

3.1. Prevalence and intensity of T. annulata infection

The figures inTable 1showT. annulata infection in fourHyalomma species engorgedon different experimental calves.

In the first experimentH. detritum andH.a. anatolicum ticks were engorged on an exper-imental calf infected withT. annulata (Sarioba), having a maximum parasitaemia of 46%.All the 34 H. detritum and 43H.a. anatolicum ticks were positive forT. annulata. Therewere significantly more infected acini inH. detritum than inH.a. anatolicum (P < 0.05).

In the second experimentH.m. marginatum andH.a. anatolicum ticks were fed on an-other experimental calf infected withT. annulata (Akdere), which developed a maximumparasitaemia of 6%. AllH.a. anatolicum ticks and 94.4% ofH.m. marginatum were pos-itive for T. annulata (Akdere). Difference between the infection prevalence rates was notsignificant (P > 0.05). There were significantly more infected acini inH.m. marginatumthan inH.a. anatolicum (P < 0.05).

In the third experimentH.a. excavatum andH.a. anatolicum ticks were engorged on anexperimental calf infected withT. annulata (Sarioba), having a maximum parasitaemia of80%. Similar proportions ofH.a. excavatum (97.1%) andH.a. anatolicum (98.8%) tickswere found to be positive forT. annulata (Sarioba). The difference between mean numbersof infected acini (mean intensity of infection) inH.a. excavatum andH.a. anatolicum wasnot significant (P > 0.05).

In the fourth experiment,H.a. excavatum, H.m. marginatum andH.a. anatolicum wereengorged on an experimental calf, infected withT. annulata (Ak-Sa), a mixture of Akdereand Sarioba GUTS stabilates, which showed a maximum parasitaemia of 87%. AllH.a.excavatum andH.a. anatolicum and 87.5% ofH.m. marginatum ticks examined for thepresence ofT. annulata were positive. The difference between the prevalence ofT. annulatainfection inH.m. marginatum and the other two species was not significant (P > 0.05). On

Table 1Comparison ofT. annulata infection in fourHyalomma species engorged on the experimental calves infected withGUTS stabilates of different origin

Experiment Hyalomma sp. Samples Infection

Size Infected Prevalence (%) Mean intensity

First H. detritum 34 34 100 241.5H.a. anatolicum 43 43 100 187.8

Second H.m. marginatum 64 61 94.4 365.2H.a. anatolicum 80 80 100 249.2

Third H.a. excavatum 157 153 97.1 274.8H.a. anatolicum 162 160 98.8 254.7

Fourth H.m. marginatum 24 22 87.5 120.9H.a. excavatum 40 40 100 195.7H.a. anatolicum 40 40 100 250.6

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Table 2Comparison ofT. annulata infection in males and females of the fourHyalomma species engorged on experimentalcalves infected withT. annulata GUTS stabilates

Hyalomma sp. Sample size Prevalence (%) Mean intensity

Total Female Male Total Female Male Total Female Male

H.a. anatolicum 325 172 153 99.3 99.4 99.3 221.8 270.9 172.2H.a. excavatum 197 114 83 97.5 96.5 98.8 273.2 318.8 227.6H.m. marginatum 88 36 52 93.2 94.4 92.3 223.2 256.6 229.9H. detritum 34 14 20 100 100 100 246.6 249.7 243.6

Table 3Comparison ofT. annulata infection inH.a. anatolicum ticks engorged on the four experimental calves infectedwith T. annulata GUTS stabilates of different origin

Theileria annulata Hyalomma anatolicum anatolicum

Origin Parasitemia (%) Sample size Infection prevalence (%) Mean intensity

Sarioba 46 43 100 187.8Sarioba 80 162 98.8 254.7Akdere 6 80 100 249.2Ak-Sa 87 40 100 250.6

the other hand there were more infected acini inH.a. anatolicum than inH.a. excavatum;but the difference between them was not significant (P > 0.05). In addition there weremore infected acini in bothH.a. excavatum andH.a. anatolicum than inH.m. marginatumand the differences between them were significant (P < 0.05).

In Table 2, it has been stated that the infection rates of four tick species ranged between94.4–100% in females and 92.3–100% in males. The infection rate difference between sexeswas found to be statistically insignificant (P > 0.05). However, in the same table it has beenpointed out that, the number of infected acini in the three tick species exceptH. detritumwas higher in females than males and this difference was found to be statistically significant(P < 0.05).

In Table 3, it has been stated that the infection rates of the only tick species,H.a.anatolicum,infected with different isolates (T.annulata (Sarioba) orT. annulata (Akdere) orT. annulata(Ak-Sa)) and fed on calves with different infection rates (6–87%) ranged between 98.8 and100% and the difference was found to be statistically insignificant (P > 0.05). Howeverit has been determined that, the infected salivary gland acini intensity in the ticks infectedwith the Sarioba isolate and fed on two calves with different parasitaemia rates was higherthan those fed on calves with higher parasitaemia rates. This difference was found to bestatistically significant (P < 0.05).

4. Discussion

This study has shown that whenH. detritum andH.a. anatolicum ticks were fed on acalf infected withT. annulata (Sarioba) and then treated identically the prevalence ofT.annulata infection were similar. However, when compared with that inH.a. anatolicum

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the mean number of infected acini perH. detritum tick was considerably higher. TheT.annulata (Sarioba) infection prevalence inH.m. marginatum and H.a. anatolicum ticksfed on another infected calf were different being lower inH.m. marginatum than inH.a.anatolicum, although the difference between the mean number of infected acini per tickin H.m. marginatum was significantly higher than inH.a. anatolicum. The percentage ofT. annulata (Akdere) infection prevalence inH.a. excavatum andH.a. anatolicum fed onanother infected calf were high and very similar to each other, whereas the differencesbetween the mean number of infected acini per tick in both species were not significantlydifferent. In a comparison ofT. annulata infection prevalences inH.m. marginatum, H.a.anatolicum andH.a. excavatum all fed on the same calf infected withT. annulata (Ak-Sa),it was observed that the percentage infection prevalences and the intensities were similar inH.a. anatolicum andH.a. excavatum but much lower inH.m. marginatum.

In female and male ticks of each of the fourHyalomma species used in the study theinfection prevalence withT. annulata did not vary markedly. The mean intensity in femaleand male ticks of allHyalomma species was different with female ticks having higherintensity than the male ticks. The sex difference was more significant inH.a. anatolicumandH.a. excavatum than inH. detritum andH.m. marginatum. On the other hand infectionprevalence did not vary markedly betweenH.a. anatolicum ticks engorged on the four calvesinfected with differentT. annulata isolates and developed parasitemia at different levels.

The determination of the pattern ofTheileria challenge in the field through tick studiesis a potentially rewarding approach toTheileria epidemiology. In the few previous studiesconducted on the role of the sex of the tick inTheileria transmission it was emphasisedby Bucher and Tangus (1986)andSangwan et al. (1989)that both the prevalence and theintensity ofTheileria infection were significantly higher in female ticks than in male ticks.However,Sangwan et al. (1989)reported that although the prevalences were very similar forfemales and males, the intensity of infection was much higher in females when comparedwith males.Walker et al. (1981)also reported significantly higher intensity in females.The relative role of male and female ticks in the transmission ofT. annulata has also beenstudied byPipano et al. (1982). He concluded that a frozen stabilate ofT. annulata preparedfrom partially engorgedH.a. excavatum females was considerably more infective, on anindividual tick basis, than a similar stabilate prepared from male ticks. Our observationscorroborate these results.

It was reported byYoung et al. (1983)that there were many more type III acini in femaleticks than in male ticks andTheileria parasites were only detected in type III acini.Scheinand Friedhoff (1978)also commented thatTheileria parasites developed mainly within cellsof type III acini. These statements demonstrate why female ticks have higherTheileriainfection prevalences than males. As stated bySangwan et al. (1989), the developmentof Theileria parasites in the vector ticks depends on several extrinsic and intrinsic factorswhich still remain to be studied. In the present study the roles of species and sex in fourHyalomma ticks and the development ofT. annulata has been clarified.

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