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Piroplasms of domestic animals in the Macedonia region of Greece 2. Piroplasms of cattle
veterinary parasitology ELSEVIER
Veterinary Parasitology 63 (1996) 57-66
Piroplasms of domestic animals in the Macedonia region of Greece 2. Piroplasms of cattle 1 Byron Papadopoulos a,*, Michel Brossard a, Nan Made Pefid b a Institut de Zoologie, Universit~ de Neuch~tel, 2007 Neuchatel, Switzerland b Institute for Infectious Diseases and Immunology, Faculty of Veterinary Medicine, PO Box 80.165, 3508 TD Utrecht, Netherlands Received 5 May 1994; accepted 1 June 1995
Abstract A study was carried out on piroplasms of cattle in the Macedonia region of Greece. During 1984-1986, 602 serum samples were collected from cattle in 33 localities. Blood smears were also prepared from 50 of the animals in 13 localities. The indirect fluorescent antibody (IFA) test revealed that 41.4% of cattle sera were positive to Theileria orientalis, 2.0% to Theileria annulata, 21.6% to Babesia bovis, 15.2% to Babesia bigemina, 5.1% to Babesia major and 2.7% to Babesia divergens. Some of the positive titres were probably due to cross reactions. T. orientalis was widely distributed. T. annulata was limited to a small number of foci. B. bigemina and B. boris, often present together, were widespread, whereas B. major was only present in a small number of localities. No convincing evidence of B. dioergens infection was found. By examination of Giemsa stained blood smears T. orientalis, T. annulata and a Babesia sp. were detected-. Keywords: Babesia spp.; Theileria spp; Cattle-Protozoa; Epidemiology-Protozoa; Greece
1. I n t r o d u c t i o n Previous reports indicate that four species of piroplasm (Theileria orientalis, Theileria annulata, Babesia bigeraina and Babesia boris) are present in Greece (Cardamatis
* Corresponding author at: Department of Biology, University of Crete, PO Box 1470, 711 10 Iraldion, Crete, Greece. ' The results of this study represent part of Ph.D. Thesis of B. Papadopoulos. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved SSDI 0 3 0 4 - 4 0 1 7 ( 9 5 ) 0 0 8 4 5 - 4
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1911, 1912; M~lanidis and Stylianopoulos, 1927; Stylianopoulos and Ananiad~s, 1933). The regions of northern Greece (Macedonia and Thrace) are considered to be endemic for piroplasmosis and particularly for theileriosis (Stylianopoulos and Ananiad~s, 1933; Cardassis, 1964). In 1942, an epidemic of tropical theileriosis with several fatal cases was described by Pavlov in a village of Macedonia (Pavlov, 1942). Theileriosis has been reported mainly in imported purebred or cross-bred cattle which seemed to be more severely affected than local animals (Cardassis, 1956, 1964). All previous studies dealt chiefly with infections diagnosed by the Public Veterinary Services, and clinical aspects and specific treatment of the diseases. There has been no previous serological survey to evaluate the distribution and the prevalence of the different piroplasms. Therefore, in our study, we have tried to define the presence, distribution and prevalence of Theileria and Babesia organisms in livestock in the Macedonia region of Greece.
2. Materials and methods
2.1. Field study area Work was done in the Macedonia region of Greece (34203 km2), which is situated between 29050'-41032 ' north and 20047'-24047 ' east (Fig. 1). This region offers a wide variety of climatic and vegetational types. Information about the inspected localities and details on the different kinds of bioclimate and vegetation present in the region is given by Papadopoulos et al. (1995a). 2.2. Experimental procedure During March-April 1984, September-October 1984, May-July 1985 (mainly) and September-October 1986, 602 serum samples were collected from cattle in 33 localities (Fig. 1). Blood smears were also prepared from 50 animals in 13 localities. During the study ticks were also collected from animals. 2.2.1. Serology The sera were tested by the indirect fluorescent antibody test (IFAT), against several piroplasm antigens: T. orientalis (Jeju), T. annulata (Bahrein), B. bigemina (Zaria), B. boris (Israel), Babesia divergens (Elspeet) and Babesia major (Ameland). The antigen strains, the antigen preparation and the IFA test have been described in detail elsewhere (Papadopoulos et al., 1995b). Negative sera gave a titre of < 80 for all antigens. However, because of some cross-reactions observed in the tests with the positive control sera against the heterologous antigens the specificity level of the test was fixed as follows: for T. orientalis at 160 (at 320 when it is associated with a titre of 1280 or higher for T. annulata); for T. annulata at 160 (at 320 when it is associated with a titre of 1280 or higher for T. orientalis); for B. bigemina at 160/320 (at 320 when it is associated with a titre of 1280 or higher for B. divergens or B. major and at 320/640 when it is associated with a titre of 1280 or higher for B. boris); for B. boris at
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160/320 (at 320 when it is associated with a titre of 1280 or higher for B. bigemina or B. divergens); for B. divergens at 160 (at320 when it is associated with a titre of 1280 or higher for B. bigemina or B. boris); for B. major at 160. Titres lower up to one dilution from the fixed specificity level were considered as doubtful, while lower titres as negative (Papadopoulos et al., 1995b).
2.2.2. Blood smears Thin blood smears were prepared from a drop of blood collected from the ear of the animal and stained by the Giemsa method. The films were air dried, fixed in methanol and stained for 1 h in a 6% dilution of Giemsa solution (Merck) in PBS pH 7.2. The smears were examined at a magnification of × 800, using an oil immersion lens.
3. Results
3.1. Serology-Vectors Species specific titres ( > 160) against T. orientalis were detected in 41.4% of examined animals and in 28 out of 33 localities surveyed (Table 1; Fig. 1). Haemaphysalis punctata, the vector of T. orientalis in Europe, was present in 17 of these 28 localities and in only one out of five localities where no animals showed positive titres against T. orientalis. In contrast, only 2.0% of examined animals had a positive reaction for T. annulata. Ten of 12 seropositive animals came from one herd which originated from a locality (Dialekto, No. 30) of low altitude in eastern Macedonia (Fig. 1). In June when the sera were collected, the herd had moved to an adjacent locality (Elafohori, No. 29) of middle altitude. Only the bioclimate of the locality of origin of the herd is favourable for Hyalomma detritum detritum, the vector of T. annulata in the Mediterranean region. However, no ticks were collected from Dialekto in spring or summer, when H. d. detritum is active. The other two seropositive animals were isolated cases from two different localities. H. d. detr/tum was not found in any locality. Specific antibodies against B. bigemina were detected in 15.2% of the cattle in 18 localities, while 21.6% of cattle in 15 localities had specific antibodies against B. bovis (Table 1; Fig. 1-). In 13 localities, cattle had specific antibodies against both B. bigemina and B. bovis. In two localities, animals with high antibody titres ( > 160) against B. boris and low titres ( 4 160) against B. bigemina were present. Animals with positive anti-B, bigemina antibody titres and doubtful or negative anti-B, boris titres were found in five localities. Boophilus annulatus, the vector of both species, was found on cattle in ten out of 13 localities which were 'positive' for both Babesia spp., as well as in two localities 'positive' for B. boris only. In the last locality where this tick was collected, one animal gave doubtful titres for both Babesia spp.. No Boophilus annulatus was found in the five localities where animals showed positive titres only against B. bigemina. Sixteen (2.7%) cattle gave positive titres (160-320) against B. divergens, but the simultaneous presence of antibodies against other Babesia spp. with higher titres in
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~,
b°A P~o .
~
~-A ,
Fig. 1. Serological results for 7". orientalis, B. bigemina and B. boris and piroplasms observed in the blood smears, by locality, in Macedonia. Legend: T. orientalis, open circle, no positive sera; open circle with centre cross, ~< 25% of the sera positive; bold circle with centre cross, 25-50% of the sera positive; solid circle, > 50% of the sera positive; B. bigemina, open diamond, no positive sera; open diamond with centre cross, ~< 25% of the sera positive; bold diamond with centre cross, 25-50% of the sera positive; solid diamond, > 50% of the sera positive; B. boris, open triangle, no positive sera; mid-bold open triangle, ~< 25% of the sera positive; bold open triangle, 25-50% of the sera positive; solid triangle, > 50% of the sera positive; solid asterisk, T. orientalis in the blood smears of cattle; open asterisk, T. annulata in the blood smears of cattle; solid star, Babesia sp. in the blood smears of a cattle.
almost all animals, suggests that most, if not all, of these were cross-reactions (Papadopoulos et al., 1995b). No correlation was found between the anti-B, divergens titres and the occurrence of its vector Ixodes ricinus, adults of which were present in 20 of the inspected localities. Antibodies against B. major were detected in 5% of the cattle. Most of the high titres against B. major were associated with high titres against B. bigemina or B. boris.
Table I Results of the IFA test of cattle sera against the different antigens Antigen
T. orientalis T. annulata B. bigemina B. bovis B. divergens B. major
Sera Total
Negative
No.
No.
%
No.
Doubtful %
No.
Positive %
601 602 592 593 592 587
247 567 325 384 479 491
41.1 94.2 54.9 64.8 80.9 83.6
105 23 177 81 97 66
17.5 3.8 29.9 11.7 16.4 11.2
249 12 90 128 16 30
41.4 2.0 15.2 21.6 2.7 5.1
No., number of sera. For the definition of negative, doubtful and positive sera see text.
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However, for 12 sera the anti-B, major titre was the highest and based on these data B. major could be present in four localities (Zervi, No. 5; Ahladohori, No. 9; Ano Apostoli, No. 13; Kalamitsi, No. 65). In these localities, as well as in four other localities (out of ten with positive titres to B. major), the animals also possessed specific antibodies against T. orientalis, which is transmitted by H. punctata also. H. punctata was found in seven of these localities. 3.2. Blood smears 3.2.1. Theileridae Theileria spp. were observed in the red blood cells of 33 out of 50 examined animals and in ten out of 13 localities where these animals were present (Fig. 1). The parasitaemia was low and never exceeded 1%. No clinical symptoms were shown by the examined animals. Considerable polymorphism of the intraerythrocytic parasites has been observed. The majority of the merozoites were elongate or short fuse-shaped, rod-shaped or club-shaped, with the exception of some animals from Elafohori (No. 29), in eastern Macedonia. Their size was between 1 and 2 /.~m. Their morphology, together with the presence of antibodies against T. orientalis, indicated that the animals belonged to this species. However, in the infected erythrocytes no bar or veil structures characteristic of T. orientalis were observed. Associated dark structures, weakly stained, resembling veils, were observed in the blood films of only two animals from Zervi (No. 5), in western Macedonia. Numerous round and oval forms, generally of small size (0.7-1.3/xm), were seen in animals from Elafohori in association with high antibody titres against T. orientalis and T. annulata, indicating mixed infections by both Theileria spp. For 43 out of 50 animals a serological test was also performed. Theilerial parasites were seen in 25 out of 30 animals which were seropositive and in six out of seven which were doubtful for T. orientalis. No parasites were observed in the six seronegative animals. 3.2.2. Babesidae Out of the total 50 animals examined, seven were suspected of having babesiosis. A single form and a double form observed in the blood film of a calf from Eleoussa (No. 51), in central Macedonia (Fig. 1), constituted the only detected parasites. Their size was similar to that of B. bovis (2 × 1 /.~m). However, the animal, which had been treated for babesiosis a week before bleeding, gave a positive serological reaction against B. bigemina.
4. Discussion
4.1. Theileridae T. orientalis, the agent of the benign theileriosis of Eurasian and Australian cattle (Morel and Uilenberg, 1981) is widespread in Macedonia, while T. annulata, the agent
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of Mediterranean or tropical theilefiosis (Uilenberg, 1981) appears to be limited to a small number of foci. T. orientalis is a cosmopolitan parasite which occurs on all continents (Uilenberg et ai., 1985). Until about 12 years ago, it was confused with T. mutans, the agent of benign African theileriosis. Morphological and serological studies have shown on the one hand the similarity between the various benign theileriae of Eurasia, Australia and America, and on the other hand their differences with T. mutans and the other African theilerias (Uilenberg et al., 1977, 1985; Minami et al., 1980) In our survey, this parasite was detected in several bovine blood smears. However, it was not possible to distinguish clearly the small bar a n d / o r the veil, intraerythrocytic structures, characteristic for this species (Uilenberg, 1981). However, we cannot be certain of the absence of these structures. In fact, relatively few parasites were seen and the time between the preparation and the staining of the smears, which was not done on the site, was sometimes very long. As no lymph node biopsy smears were made, no schizonts were seen. It is only recently that Uilenberg et al. (1985) and Minami et al. (1990) succeeded in demonstrating the schizont stage of the parasite in cattle infected experimentally by ticks with strains from Korea and Japan, respectively. Piroplasms were detected in 31 out of 37 cattle which gave a positive or doubtful serological reaction and blood smears of these were also prepared. It appears that the animals remain carriers. This confirms previous work where animals recovered from a T. orientalis infection retained significant levels of antibody titres for 22 months. During this time parasites were detectable in blood smears (Morzaria et al., 1977). T. orientalis does not appear to be pathogenic to cattle in Macedonia. Moreover, we have no reports on outbreaks or clinical cases due to this species in Greece. With the exception of some regions in East Asia, infections of T. orientalis are inapparent or subclinical (Brocklesby et al., 1972; Hun, 1978; Minami et al., 1981; Uilenberg and Hashemi-Fesharki, 1984; Uilenberg et al., 1985). All proven vectors of T. orientalis belong to the genus Haemaphysalis (Uilenberg, 1981). H. punctata has been shown to be the vector of the parasite in the UK, the Netherlands and Germany (Brocklesby and Barnett, 1972; Mohammed, 1976; Liebisch and Rahman, 1978). This tick species is quite frequent in Macedonia, but no immature stages have been collected from cattle in this region (Papadopoulos et al., 1995a). As Theileria spp. are transmitted only trans-stadially, infestation of cattle by larvae and nymphs is necessary for the accomplishment of the cycle of the parasite. In Greece, another tick species could act as a vector of T. orientalis. The wide distribution of the parasite and its high prevalence in the older animals indicate that the vector must be an abundant tick species. We suspect that it may be Rhipicephalus bursa, a monotropic two-host species, present throughout Macedonia (Papadopoulos et al., 1995a). Different Theileria spp. are transmitted by ticks of the genus Rhipicephalus (Uilenberg, 1981; Young, 1981). The Turkish strain of T. ovis is transmitted by R. bursa (Wamecke, 1978), while Theileria separata, another theileria of small ruminants, is transmitted by Rhipicephalus eoertsi, which belongs to the same subgenus Digineus (Mehlhorn et al., 1979, Uilenberg, 1981). While Cardassis (1964) reported that T. annulata was endemic in central and eastern Macedonia, this is not supported by our study. The only herd in which several animals had specific anti-T, annulata antibodies, originated from Dialekto (No. 30), a village at
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the foot of a mountainous chain in eastern Macedonia. Clinical cases of theileriosis are still recorded at the foot of this mountainous chain. Except for two other animals from two separate localities, no other cattle had an antibody titre against T. annulata. Moreover, we did not obtain from the Public Veterinary Stations any reliable indication about clinical theileriosis cases in other regions of Macedonia. We suggest that the number of foci of tropical theileriosis has dramatically decreased. The extension of the big cities and the tourist development on the coasts in the second half of this century have limited the pastures available for the cattle. Zero-grazing becomes more and more frequent, but the animals continue to be put out to pasture in regions of middle or high altitude. The climate of these regions is not favourable for H. d. detritum, vector of T. annulata in southern Europe. The normal distribution of this tick is located at the level of warm Mediterranean maquis (Morel, 1965). Nevertheless, H. d. detritum, whose vector role has been demonstrated for the first time in Algeria (Sergent et al., 1928), remains a candidate for the transmission of T. annulata in Greece. The other species of Hyalomma present in Macedonia cannot play a role in the natural transmission of T. annulata, although they have been shown to be experimental vectors of this parasite (Delpy, 1949). Neither the ditropic three-host cycle of Hyalomma anatolicum excavaturn, nor the ditropic two-host cycle of Hyalorama marginamm marginatum, nor the monotropic one-host cycle of Hyalomma detritum scupense, can maintain the exclusively ~rans-stadial transmission of a Theileria sp. The absence or scarcity of H. d. detritura in the biotopes where cattle presently graze, could explain the decreasing incidence of tropical theileriosis. 4.2. Babesidae In Macedonia, B. bigemina and B. bovis are present in several localities, and often coexist. B. bigemina is more widely spread than B. bovis. The transmission of B. bovis seems to be less frequent, as is the case in Australia, where ticks infected by B. boris are much more rare than ticks infected by B. bigemina (Johnston, 1967; Mahoney and Mirre, 1971). Thus, in enzootic regions, B. bigemina infection develops generally before the B. bovis infection (Mahoney, 1962). The inoculation rate is higher for B. bigemina (Mahoney, 1969), but the parasitaemia of B. boris lasts longer (Mahoney, 1969; Mahoney et al., 1973). It is likely that the anti-B, bovis antibodies are detectable for longer periods because of the persistence of the parasite in the animal. Thus, a higher number of animals seropositive to B. boris was found in certain localities where both parasites were present. In Greece, infections due to B. bovis constitute the most common clinical babesiosis of cattle. This is an acute and very severe infection. On the contrary, babesiosis due to B. bigemina is much less frequent and it is rarely acute (Stylianopoulos and Ananiadts, 1933). In all the southern and eastern part of Serbia the situation is similar. B. boris is the agent responsible for more then 90% of clinical cases of piroplasmosis (Simi~ and Neveni~, 1953; Mekuli, 1959). The Public Veterinary Services do not perform any specific diagnostic test for piroplasms. However, the forms of Babesia sp. observed in the blood film of a calf, treated for piroplasmosis some days previously, were of a small size (about 2 /~m).
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Both parasites share the same vectors, namely, species of the genus Boophilus. Transovarian transmission of B. bigemina and B. bovis by the tick Boophilus annulatus has been demonstrated experimentally (Hadani et al., 1974). In Macedonia, Boophilus annulatus was collected from the majority of localities where cattle possessed antibodies against both parasites or against B. boris only. The absence of this tick species from the localities where cattle showed specific serologic reactions only to B. bigemina, leads us to assume that it could be also transmitted by another tick species. Our serological study did not provide any evidence for the occurrence of B. divergens in Macedonia. Serological studies conducted in different parts of Europe showed that this infection is quite frequent in cattle (Adam and Blewett, 1978; Weiland et al., 1980; Taylor et al., 1982; Gem and Brossard, 1986; Christensson and Mor6n, 1987). B. divergens is transmitted by lxodes ricinus (Joyner et al., 1963), Adults of this species often parasitised the cattle in Macedonia, while larvae or nymphs have never been collected from livestock in the region (Papadopoulos et ai., 1995a). Although all stages of I. ricinus have been shown experimentally to be capable of transmitting the infection to bovine hosts (Donelly and Peirce, 1975), in the field the transmission of B. divergens is assumed to mainly recur with nymphs, and the role of female ticks in the transmission of the infection under natural conditions is actually contested (Morel, 1980). The absence of infections due to B. divergens in Macedonia probably comes from the different behaviour of immature stages of I. ricinus among Greece and temperate Europe. B. major, like B. divergens, has never been reported before from Greece, but its presence is known in certain countries of temperate Europe (Bool, 1961; Brocklesby and Barnett, 1970; Liebisch et al., 1976). The serological survey showed that B. major is rare in Macedonia, in spite of the presence of H. punctata, vector of this piroplasm (Brocklesby and Barnett, 1970), in a large part of the sampled localities. Immature stages of the vector are absent from cattle in the study area (Papadopoulos et al., 1995a) and this could partly explain the scarcity of this infection.
Acknowledgements We wish to acknowledge the valuable assistance of the public veterinary practitioners in the region of Macedonia. We are grateful to the farmers who readily allowed us to bleed their herds. Our thanks are also due to F.F.J. Franssen and M. de Groot for technical assistance in serology. Finally, we are indebted to Prof. G. Uilenberg for reviewing the manuscript. This work was partially financed by Diagnostic Parasitaire (Institut de Zoologie), Neuch,~tei, Switzerland.
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Booi, P.H., 1961. De Babesia-soorten van her fund in Nederland: Babesia divergens en Babesia major. Tijdschr. Diergeneesk., 86: 28-37. Brocklesby, D.W. and Barnett S.F., 1970. Large Babesia species transmitted to splenectomized calves by field collections of British ticks (Haemaphysalis punctata). Nature, 228 (5277): 1215. Brocklesby, D.W. and Barnett S.F., 1972. The tick Haemaphysalis punctata shown to be a vector of Theileria mutans in Britain. Vet. Roe., 90:512. Brocklesby, D.W., Sellwood, S.A. and Harness, E., 1972. Some characteristics of a strain of Theileria mutans (Tbeiler, 1906) isolated from cattle in the County of Kent, England, and maintained in splenectomiscd calves. Int. J. Parasitol., 2: 265-271. Cardamatis, J.P., 1911. Des Piroplasmiases et leishmaniases. Zbl. Bakt., Abt.I.Orig., 60 (6): 511-523. Cardamatis, J.P., 1912. Piroplasmoses des hnvid~s en Grace. Bull. Soc. Path. Exot., 5: 87-88. Cardassis, J., 1956. Essais de taltement de la theii~riose bovine grecque par quelques antipaludiques de synth~se: la quinacrine et la paludrine. Bull. Acad. V~t. pr., 39: 66-72. Cardassis, J., 1964. Epi tis Theileria annulata ofilomenis theileriaseos ton vooidon en Helladi. 1. Epizootiologia ke kliniki meleti. Elliniki Ktiniatriki, 7 (2): 77-90, (in Greek, with French abstract). Christensson, D.A. and Mor~n, T., 1987. Seroresponse (IgG) after vaccination and natural infection of cattle with Babesia divergens. Acta Vet. Scand., 28 (3-4): 393-402. Delpy, L,P., 1949. Recherches effectu~,es en Iran sur Theileria annalata Dschunkowsky et Luhz, et sa transmission clans les conditions naturelles et exp~rimentales. Bull. Soc. Path. Exot., 42: 285-294. Donnelly, J. and Peirce, M.A., 1975. Experiments on the transmission of Babesia divergens to cattle by the tick lxodes ricinus. Int. J. Parasit., 5: 363-367. Gem, L. and Brossard, M., 1986. Evolution annnelle de l'infestation de bovins par la tlque lxodes ricinus L. et de l'infection de ces ectoparasites par Babesia divergens dans le Clos-du Donbs (Jura, Suisse). Schweiz. Arch. Tierbeilk., 128: 361-363. Hadani, A., Pipano, E., Tsafrir, N., Rauchbach, K. and Mayer, E., 1974. The transmission of Babesia bigemina, Babesiella berbera and Anaplasma centrale by Boophilus annulatus (Say). Refuah Vet., 31 (4): 149-154. Han, T.W., 1978. Studies on bovine theileriosis in Korea. Res. Rep. Off. Rural Dev., 20: 53-88. Johnston, L.A.Y., 1967. Epidemiology of bovine babesiosis in Northern Queensland. Aust. Vet. J., 43: 427-432. Joyner, L.P., Payne, R.C., Takahashi, K., Brocklesby, D.W. and Irvin, A.D., 1979. Serological comparison of British Theileria mutans and Japanese T. sergenti. Res. Vet. Sci., 26: 387-388. Liebisch and Rahman, 1978. Occurrence of Haemaphysalis punctata, the vector of Babesia major and Theileria mutanz, in cattle in North Germany. In: J.K.H. Wilde (Editor), Tick-borne diseases and their vectors. Proceedings of an International Conference, Edinburgh, 1976. University Press, Edinburgh, pp. 188-191. Liebisch, A., Melfsen, J. and Rahman, M.S., 1976. Zum vorkommen der Zecke Haemaphysalispunctata (Can. et Fanz., 1877) und yon Babesia major beim rind in NoKldeutschland. Berl. Miinch. Tierarztl. Wschr., 89: 477-480. Mahoney, D.F., 1962. The epidemiology of babesiosis in cattle. Aust. Vet. J., 24 (7): 310-313. Mahuney, D.F., 1969. Bovine babesiasis: a study of factors concerned in transmission. Ann. Trop. Med. Parasitol, 63 (1): 1-14. Mahoney, D.F. and Mirre, G.B., 1971. Bovine babesiasis: estimation of infection rates in the tick vector Boophilus microplus. Ann, Trop. Med. Parasitol., 65:309-317. Mahoney, D.F., Wright, I.G. and Mine, G.B., 1973. Bovine babesiasis: The persistence of immunity to Babesia argentina and B. bigemina in calves (Bos taurus) after naturally acquired infection. Ann. Trop. Med. Parasit., 67 (2): 197-203. Mehlhorn, H., Schein, E. and Wamecke, M., 1979. Electron microscopic studies on Theileria ovis: development of kinetics in the gut of the vector tick, Rhipicephalus evertsi evertsi, and their transformation within cells of the salivary glands. J. Protozool., 26: 377-385. Mekuli, E., 1959. Contribution ~ la connalssance des piroplasmoses des animaux domestiques h Kosovo et Metohie. Acta Vet., 9 (4): 103-112. (In Serbian, with French abstrac0. M61anidis, C. and Stylianoponlos, M., 1927. Notes sur la peste bovine en Grb.ce. Rev. G6n. M6d. V6t., 36: 72-78.
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Minami, T., Fusinaga, T., Fumya, K. and lshihara, T., 1980. Clinicohematologic and serological comparison of Japanese and Russian strains of Theileria sergenti. Nat. Inst. Anim. Health Q., 20: 44-52. Minami, T., ishihara, T. and Fujita, J., 1981. Bovine theileriosis and its control in Japan. In: A.D. lrvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff Publishers, The Hague, pp. 94-96. Minami, T., Kawazu, S., Shimura, K., Fujisaki, K., Kamio, T. and Ito, S., 1990. Detection of the schizont stage in experimentally infected cattle with Japanese Theileria sergenti. Jpn. J. Vet. Sci., 52 (3): 601-604. Mohammed, A.N., 1976. Prevalence and experimental transmission of bovine piroplasmosis in the Netherlands. Tijdschr. Diergeneesk., 101 (8): 413-420. Morel, P.C., 1965. Les tiques d'Afrique et du Bassin M&literranren (Ixodoidea). Unpublished manuscript, 695 Pp. Morel, P.C., 1980. Rrflexions sur l'rpizootiologie de la babrsiose bovine europrenne ~ Babesia divergens. Bull. Acad. Vrt. France, 53: 451-456. Morel, P.C. and Uilenberg, G., 1981. Sur la nomenclature de quelques Theileria (Sporozoa, Babesioidea) des ruminants domestiques. Rev. I~lev. Mrd. Vrt. Pays Trop., 34 (2): 139-143. Morzaria, S.P., Brocklesby, D.W. and Harradine, D.L., 1977. Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain. Vet. Rev., 100: 484-487. Papadopoulos, B., Morel, P.-C. and Aeschlimann, A., 1995a. Ticks of domestic animals in the Macedonia region of Greece. Vet. Parasitol., 63: 25-40. Papadopoulos, B., Perir, N.M. and Uilenberg, G., 1995b. Piroplasms of domestic animals in the Macedonia region Greece. 1. Serological cross-reactions. Vet. Parasitol., 63: 41-56. Pavlov, P., 1942. Das Vorkommen yon Theileriose in Mazedonien. Dtsch. Tiefftrztl. Wschr., 40 (43-44): 458-460. Sergent, E., Donatien, A.L., Parrot, L.M. and Lestoquard, F., 1928. Transmission de la piroplasmose bovine Theileria dispar, de l'Afrique du Nord, par la tique Hyalomma mauritanicum. C.R. Hebd. Sranc. Acad. Sei., 187 (8): 259-260. Simir, (~.P. and NevenitS, V., 1953. Babesiella boois (Bab~s, 1888) et Babesiella berbera Sergent, Donatien, Parrot, Plantureux et Rougebief, 1924 sont-ils synonymes? Arch. Inst. Pasteur Algrr., 31 (2): 91-101. Stylianopoulos, M. and Ananiad~s, B., 1933. Contribution ~ l'~tude des piroplasmoses en Grb,ce. IV. Les piroplasmoses bovines. Bull. Soc. Path. Exot., 26 (9): 1153-1156. Taylor, S.M., Kenny, J. and Strain, A., 1982. The distribution of Babesia divergens infection within the cattle population of Northern Ireland. Br. Vct. J., 138: 384-392. Uilenberg, G., 1981. Theilerial species of domestic livestock. In: A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Ni~off Publishers, The Hague, pp. 4-37. Uilenberg, G. and Hashemi-Fersharki, R., 1984. Theileria orientalis in Iran. Vet. Q., 6 (l): 1-4. Uilenberg, G., Mpangala, C., Mcgregor, W. and Callow, L.L., 1977. Biological differences between African Theileria mutans (Theiler 1906) and two benign species of Theileria of cattle in Australia and Britain. Aust. Vet. J., 53: 271-273. Uilenberg, G., Perir, N.M., Spanjer, A.A.M. and Franssen, F.F.J., 1985. Theileria orientalis, a cosmopolitan blood parasite of cattle: demonstration of the schizont stage. Res. Vet. Sci., 38: 352-360. Young, A.S., 1981. The epidemiology of theileriosis in East Africa. In: A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Ni~off Publishers, The Hague, pp. 38-55. Wamecke, M., 1978. A survey of the infestation rate of ixodid ticks with piroplasms in Turkey. In: J.K.H. Wilde (Editor), Tick-Borne Diseases and their Vectors. Proceedings of an International Conference, Edinburgh, 1976. University Press, Edinburgh, pp. 166-167. Weiland, G., Reif, L., Schmidt, M. and Boch, J., 1980. Serologische Untersuchungen zum Nachweis der Babesia divergens-lnfektion des Rindes. Berl. Miinch. TierSaxtl. Wsehr., 93: 261-264.
Veterinary Parasitology 63 (1996) 57-66
Piroplasms of domestic animals in the Macedonia region of Greece 2. Piroplasms of cattle 1 Byron Papadopoulos a,*, Michel Brossard a, Nan Made Pefid b a Institut de Zoologie, Universit~ de Neuch~tel, 2007 Neuchatel, Switzerland b Institute for Infectious Diseases and Immunology, Faculty of Veterinary Medicine, PO Box 80.165, 3508 TD Utrecht, Netherlands Received 5 May 1994; accepted 1 June 1995
Abstract A study was carried out on piroplasms of cattle in the Macedonia region of Greece. During 1984-1986, 602 serum samples were collected from cattle in 33 localities. Blood smears were also prepared from 50 of the animals in 13 localities. The indirect fluorescent antibody (IFA) test revealed that 41.4% of cattle sera were positive to Theileria orientalis, 2.0% to Theileria annulata, 21.6% to Babesia bovis, 15.2% to Babesia bigemina, 5.1% to Babesia major and 2.7% to Babesia divergens. Some of the positive titres were probably due to cross reactions. T. orientalis was widely distributed. T. annulata was limited to a small number of foci. B. bigemina and B. boris, often present together, were widespread, whereas B. major was only present in a small number of localities. No convincing evidence of B. dioergens infection was found. By examination of Giemsa stained blood smears T. orientalis, T. annulata and a Babesia sp. were detected-. Keywords: Babesia spp.; Theileria spp; Cattle-Protozoa; Epidemiology-Protozoa; Greece
1. I n t r o d u c t i o n Previous reports indicate that four species of piroplasm (Theileria orientalis, Theileria annulata, Babesia bigeraina and Babesia boris) are present in Greece (Cardamatis
* Corresponding author at: Department of Biology, University of Crete, PO Box 1470, 711 10 Iraldion, Crete, Greece. ' The results of this study represent part of Ph.D. Thesis of B. Papadopoulos. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved SSDI 0 3 0 4 - 4 0 1 7 ( 9 5 ) 0 0 8 4 5 - 4
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1911, 1912; M~lanidis and Stylianopoulos, 1927; Stylianopoulos and Ananiad~s, 1933). The regions of northern Greece (Macedonia and Thrace) are considered to be endemic for piroplasmosis and particularly for theileriosis (Stylianopoulos and Ananiad~s, 1933; Cardassis, 1964). In 1942, an epidemic of tropical theileriosis with several fatal cases was described by Pavlov in a village of Macedonia (Pavlov, 1942). Theileriosis has been reported mainly in imported purebred or cross-bred cattle which seemed to be more severely affected than local animals (Cardassis, 1956, 1964). All previous studies dealt chiefly with infections diagnosed by the Public Veterinary Services, and clinical aspects and specific treatment of the diseases. There has been no previous serological survey to evaluate the distribution and the prevalence of the different piroplasms. Therefore, in our study, we have tried to define the presence, distribution and prevalence of Theileria and Babesia organisms in livestock in the Macedonia region of Greece.
2. Materials and methods
2.1. Field study area Work was done in the Macedonia region of Greece (34203 km2), which is situated between 29050'-41032 ' north and 20047'-24047 ' east (Fig. 1). This region offers a wide variety of climatic and vegetational types. Information about the inspected localities and details on the different kinds of bioclimate and vegetation present in the region is given by Papadopoulos et al. (1995a). 2.2. Experimental procedure During March-April 1984, September-October 1984, May-July 1985 (mainly) and September-October 1986, 602 serum samples were collected from cattle in 33 localities (Fig. 1). Blood smears were also prepared from 50 animals in 13 localities. During the study ticks were also collected from animals. 2.2.1. Serology The sera were tested by the indirect fluorescent antibody test (IFAT), against several piroplasm antigens: T. orientalis (Jeju), T. annulata (Bahrein), B. bigemina (Zaria), B. boris (Israel), Babesia divergens (Elspeet) and Babesia major (Ameland). The antigen strains, the antigen preparation and the IFA test have been described in detail elsewhere (Papadopoulos et al., 1995b). Negative sera gave a titre of < 80 for all antigens. However, because of some cross-reactions observed in the tests with the positive control sera against the heterologous antigens the specificity level of the test was fixed as follows: for T. orientalis at 160 (at 320 when it is associated with a titre of 1280 or higher for T. annulata); for T. annulata at 160 (at 320 when it is associated with a titre of 1280 or higher for T. orientalis); for B. bigemina at 160/320 (at 320 when it is associated with a titre of 1280 or higher for B. divergens or B. major and at 320/640 when it is associated with a titre of 1280 or higher for B. boris); for B. boris at
B. Papadopoulos et al./Veterinary Parasitology 63 (1996) 57-66
59
160/320 (at 320 when it is associated with a titre of 1280 or higher for B. bigemina or B. divergens); for B. divergens at 160 (at320 when it is associated with a titre of 1280 or higher for B. bigemina or B. boris); for B. major at 160. Titres lower up to one dilution from the fixed specificity level were considered as doubtful, while lower titres as negative (Papadopoulos et al., 1995b).
2.2.2. Blood smears Thin blood smears were prepared from a drop of blood collected from the ear of the animal and stained by the Giemsa method. The films were air dried, fixed in methanol and stained for 1 h in a 6% dilution of Giemsa solution (Merck) in PBS pH 7.2. The smears were examined at a magnification of × 800, using an oil immersion lens.
3. Results
3.1. Serology-Vectors Species specific titres ( > 160) against T. orientalis were detected in 41.4% of examined animals and in 28 out of 33 localities surveyed (Table 1; Fig. 1). Haemaphysalis punctata, the vector of T. orientalis in Europe, was present in 17 of these 28 localities and in only one out of five localities where no animals showed positive titres against T. orientalis. In contrast, only 2.0% of examined animals had a positive reaction for T. annulata. Ten of 12 seropositive animals came from one herd which originated from a locality (Dialekto, No. 30) of low altitude in eastern Macedonia (Fig. 1). In June when the sera were collected, the herd had moved to an adjacent locality (Elafohori, No. 29) of middle altitude. Only the bioclimate of the locality of origin of the herd is favourable for Hyalomma detritum detritum, the vector of T. annulata in the Mediterranean region. However, no ticks were collected from Dialekto in spring or summer, when H. d. detritum is active. The other two seropositive animals were isolated cases from two different localities. H. d. detr/tum was not found in any locality. Specific antibodies against B. bigemina were detected in 15.2% of the cattle in 18 localities, while 21.6% of cattle in 15 localities had specific antibodies against B. bovis (Table 1; Fig. 1-). In 13 localities, cattle had specific antibodies against both B. bigemina and B. bovis. In two localities, animals with high antibody titres ( > 160) against B. boris and low titres ( 4 160) against B. bigemina were present. Animals with positive anti-B, bigemina antibody titres and doubtful or negative anti-B, boris titres were found in five localities. Boophilus annulatus, the vector of both species, was found on cattle in ten out of 13 localities which were 'positive' for both Babesia spp., as well as in two localities 'positive' for B. boris only. In the last locality where this tick was collected, one animal gave doubtful titres for both Babesia spp.. No Boophilus annulatus was found in the five localities where animals showed positive titres only against B. bigemina. Sixteen (2.7%) cattle gave positive titres (160-320) against B. divergens, but the simultaneous presence of antibodies against other Babesia spp. with higher titres in
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B. Papadopoulos et al. / Veterinary Parasitology 63 (1996) 5 7 - 6 6
~,
b°A P~o .
~
~-A ,
Fig. 1. Serological results for 7". orientalis, B. bigemina and B. boris and piroplasms observed in the blood smears, by locality, in Macedonia. Legend: T. orientalis, open circle, no positive sera; open circle with centre cross, ~< 25% of the sera positive; bold circle with centre cross, 25-50% of the sera positive; solid circle, > 50% of the sera positive; B. bigemina, open diamond, no positive sera; open diamond with centre cross, ~< 25% of the sera positive; bold diamond with centre cross, 25-50% of the sera positive; solid diamond, > 50% of the sera positive; B. boris, open triangle, no positive sera; mid-bold open triangle, ~< 25% of the sera positive; bold open triangle, 25-50% of the sera positive; solid triangle, > 50% of the sera positive; solid asterisk, T. orientalis in the blood smears of cattle; open asterisk, T. annulata in the blood smears of cattle; solid star, Babesia sp. in the blood smears of a cattle.
almost all animals, suggests that most, if not all, of these were cross-reactions (Papadopoulos et al., 1995b). No correlation was found between the anti-B, divergens titres and the occurrence of its vector Ixodes ricinus, adults of which were present in 20 of the inspected localities. Antibodies against B. major were detected in 5% of the cattle. Most of the high titres against B. major were associated with high titres against B. bigemina or B. boris.
Table I Results of the IFA test of cattle sera against the different antigens Antigen
T. orientalis T. annulata B. bigemina B. bovis B. divergens B. major
Sera Total
Negative
No.
No.
%
No.
Doubtful %
No.
Positive %
601 602 592 593 592 587
247 567 325 384 479 491
41.1 94.2 54.9 64.8 80.9 83.6
105 23 177 81 97 66
17.5 3.8 29.9 11.7 16.4 11.2
249 12 90 128 16 30
41.4 2.0 15.2 21.6 2.7 5.1
No., number of sera. For the definition of negative, doubtful and positive sera see text.
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However, for 12 sera the anti-B, major titre was the highest and based on these data B. major could be present in four localities (Zervi, No. 5; Ahladohori, No. 9; Ano Apostoli, No. 13; Kalamitsi, No. 65). In these localities, as well as in four other localities (out of ten with positive titres to B. major), the animals also possessed specific antibodies against T. orientalis, which is transmitted by H. punctata also. H. punctata was found in seven of these localities. 3.2. Blood smears 3.2.1. Theileridae Theileria spp. were observed in the red blood cells of 33 out of 50 examined animals and in ten out of 13 localities where these animals were present (Fig. 1). The parasitaemia was low and never exceeded 1%. No clinical symptoms were shown by the examined animals. Considerable polymorphism of the intraerythrocytic parasites has been observed. The majority of the merozoites were elongate or short fuse-shaped, rod-shaped or club-shaped, with the exception of some animals from Elafohori (No. 29), in eastern Macedonia. Their size was between 1 and 2 /.~m. Their morphology, together with the presence of antibodies against T. orientalis, indicated that the animals belonged to this species. However, in the infected erythrocytes no bar or veil structures characteristic of T. orientalis were observed. Associated dark structures, weakly stained, resembling veils, were observed in the blood films of only two animals from Zervi (No. 5), in western Macedonia. Numerous round and oval forms, generally of small size (0.7-1.3/xm), were seen in animals from Elafohori in association with high antibody titres against T. orientalis and T. annulata, indicating mixed infections by both Theileria spp. For 43 out of 50 animals a serological test was also performed. Theilerial parasites were seen in 25 out of 30 animals which were seropositive and in six out of seven which were doubtful for T. orientalis. No parasites were observed in the six seronegative animals. 3.2.2. Babesidae Out of the total 50 animals examined, seven were suspected of having babesiosis. A single form and a double form observed in the blood film of a calf from Eleoussa (No. 51), in central Macedonia (Fig. 1), constituted the only detected parasites. Their size was similar to that of B. bovis (2 × 1 /.~m). However, the animal, which had been treated for babesiosis a week before bleeding, gave a positive serological reaction against B. bigemina.
4. Discussion
4.1. Theileridae T. orientalis, the agent of the benign theileriosis of Eurasian and Australian cattle (Morel and Uilenberg, 1981) is widespread in Macedonia, while T. annulata, the agent
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B. Papadopoulos et al./ Veterinary Parasitology 63 (1996) 57-66
of Mediterranean or tropical theilefiosis (Uilenberg, 1981) appears to be limited to a small number of foci. T. orientalis is a cosmopolitan parasite which occurs on all continents (Uilenberg et ai., 1985). Until about 12 years ago, it was confused with T. mutans, the agent of benign African theileriosis. Morphological and serological studies have shown on the one hand the similarity between the various benign theileriae of Eurasia, Australia and America, and on the other hand their differences with T. mutans and the other African theilerias (Uilenberg et al., 1977, 1985; Minami et al., 1980) In our survey, this parasite was detected in several bovine blood smears. However, it was not possible to distinguish clearly the small bar a n d / o r the veil, intraerythrocytic structures, characteristic for this species (Uilenberg, 1981). However, we cannot be certain of the absence of these structures. In fact, relatively few parasites were seen and the time between the preparation and the staining of the smears, which was not done on the site, was sometimes very long. As no lymph node biopsy smears were made, no schizonts were seen. It is only recently that Uilenberg et al. (1985) and Minami et al. (1990) succeeded in demonstrating the schizont stage of the parasite in cattle infected experimentally by ticks with strains from Korea and Japan, respectively. Piroplasms were detected in 31 out of 37 cattle which gave a positive or doubtful serological reaction and blood smears of these were also prepared. It appears that the animals remain carriers. This confirms previous work where animals recovered from a T. orientalis infection retained significant levels of antibody titres for 22 months. During this time parasites were detectable in blood smears (Morzaria et al., 1977). T. orientalis does not appear to be pathogenic to cattle in Macedonia. Moreover, we have no reports on outbreaks or clinical cases due to this species in Greece. With the exception of some regions in East Asia, infections of T. orientalis are inapparent or subclinical (Brocklesby et al., 1972; Hun, 1978; Minami et al., 1981; Uilenberg and Hashemi-Fesharki, 1984; Uilenberg et al., 1985). All proven vectors of T. orientalis belong to the genus Haemaphysalis (Uilenberg, 1981). H. punctata has been shown to be the vector of the parasite in the UK, the Netherlands and Germany (Brocklesby and Barnett, 1972; Mohammed, 1976; Liebisch and Rahman, 1978). This tick species is quite frequent in Macedonia, but no immature stages have been collected from cattle in this region (Papadopoulos et al., 1995a). As Theileria spp. are transmitted only trans-stadially, infestation of cattle by larvae and nymphs is necessary for the accomplishment of the cycle of the parasite. In Greece, another tick species could act as a vector of T. orientalis. The wide distribution of the parasite and its high prevalence in the older animals indicate that the vector must be an abundant tick species. We suspect that it may be Rhipicephalus bursa, a monotropic two-host species, present throughout Macedonia (Papadopoulos et al., 1995a). Different Theileria spp. are transmitted by ticks of the genus Rhipicephalus (Uilenberg, 1981; Young, 1981). The Turkish strain of T. ovis is transmitted by R. bursa (Wamecke, 1978), while Theileria separata, another theileria of small ruminants, is transmitted by Rhipicephalus eoertsi, which belongs to the same subgenus Digineus (Mehlhorn et al., 1979, Uilenberg, 1981). While Cardassis (1964) reported that T. annulata was endemic in central and eastern Macedonia, this is not supported by our study. The only herd in which several animals had specific anti-T, annulata antibodies, originated from Dialekto (No. 30), a village at
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the foot of a mountainous chain in eastern Macedonia. Clinical cases of theileriosis are still recorded at the foot of this mountainous chain. Except for two other animals from two separate localities, no other cattle had an antibody titre against T. annulata. Moreover, we did not obtain from the Public Veterinary Stations any reliable indication about clinical theileriosis cases in other regions of Macedonia. We suggest that the number of foci of tropical theileriosis has dramatically decreased. The extension of the big cities and the tourist development on the coasts in the second half of this century have limited the pastures available for the cattle. Zero-grazing becomes more and more frequent, but the animals continue to be put out to pasture in regions of middle or high altitude. The climate of these regions is not favourable for H. d. detritum, vector of T. annulata in southern Europe. The normal distribution of this tick is located at the level of warm Mediterranean maquis (Morel, 1965). Nevertheless, H. d. detritum, whose vector role has been demonstrated for the first time in Algeria (Sergent et al., 1928), remains a candidate for the transmission of T. annulata in Greece. The other species of Hyalomma present in Macedonia cannot play a role in the natural transmission of T. annulata, although they have been shown to be experimental vectors of this parasite (Delpy, 1949). Neither the ditropic three-host cycle of Hyalomma anatolicum excavaturn, nor the ditropic two-host cycle of Hyalorama marginamm marginatum, nor the monotropic one-host cycle of Hyalomma detritum scupense, can maintain the exclusively ~rans-stadial transmission of a Theileria sp. The absence or scarcity of H. d. detritura in the biotopes where cattle presently graze, could explain the decreasing incidence of tropical theileriosis. 4.2. Babesidae In Macedonia, B. bigemina and B. bovis are present in several localities, and often coexist. B. bigemina is more widely spread than B. bovis. The transmission of B. bovis seems to be less frequent, as is the case in Australia, where ticks infected by B. boris are much more rare than ticks infected by B. bigemina (Johnston, 1967; Mahoney and Mirre, 1971). Thus, in enzootic regions, B. bigemina infection develops generally before the B. bovis infection (Mahoney, 1962). The inoculation rate is higher for B. bigemina (Mahoney, 1969), but the parasitaemia of B. boris lasts longer (Mahoney, 1969; Mahoney et al., 1973). It is likely that the anti-B, bovis antibodies are detectable for longer periods because of the persistence of the parasite in the animal. Thus, a higher number of animals seropositive to B. boris was found in certain localities where both parasites were present. In Greece, infections due to B. bovis constitute the most common clinical babesiosis of cattle. This is an acute and very severe infection. On the contrary, babesiosis due to B. bigemina is much less frequent and it is rarely acute (Stylianopoulos and Ananiadts, 1933). In all the southern and eastern part of Serbia the situation is similar. B. boris is the agent responsible for more then 90% of clinical cases of piroplasmosis (Simi~ and Neveni~, 1953; Mekuli, 1959). The Public Veterinary Services do not perform any specific diagnostic test for piroplasms. However, the forms of Babesia sp. observed in the blood film of a calf, treated for piroplasmosis some days previously, were of a small size (about 2 /~m).
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Both parasites share the same vectors, namely, species of the genus Boophilus. Transovarian transmission of B. bigemina and B. bovis by the tick Boophilus annulatus has been demonstrated experimentally (Hadani et al., 1974). In Macedonia, Boophilus annulatus was collected from the majority of localities where cattle possessed antibodies against both parasites or against B. boris only. The absence of this tick species from the localities where cattle showed specific serologic reactions only to B. bigemina, leads us to assume that it could be also transmitted by another tick species. Our serological study did not provide any evidence for the occurrence of B. divergens in Macedonia. Serological studies conducted in different parts of Europe showed that this infection is quite frequent in cattle (Adam and Blewett, 1978; Weiland et al., 1980; Taylor et al., 1982; Gem and Brossard, 1986; Christensson and Mor6n, 1987). B. divergens is transmitted by lxodes ricinus (Joyner et al., 1963), Adults of this species often parasitised the cattle in Macedonia, while larvae or nymphs have never been collected from livestock in the region (Papadopoulos et ai., 1995a). Although all stages of I. ricinus have been shown experimentally to be capable of transmitting the infection to bovine hosts (Donelly and Peirce, 1975), in the field the transmission of B. divergens is assumed to mainly recur with nymphs, and the role of female ticks in the transmission of the infection under natural conditions is actually contested (Morel, 1980). The absence of infections due to B. divergens in Macedonia probably comes from the different behaviour of immature stages of I. ricinus among Greece and temperate Europe. B. major, like B. divergens, has never been reported before from Greece, but its presence is known in certain countries of temperate Europe (Bool, 1961; Brocklesby and Barnett, 1970; Liebisch et al., 1976). The serological survey showed that B. major is rare in Macedonia, in spite of the presence of H. punctata, vector of this piroplasm (Brocklesby and Barnett, 1970), in a large part of the sampled localities. Immature stages of the vector are absent from cattle in the study area (Papadopoulos et al., 1995a) and this could partly explain the scarcity of this infection.
Acknowledgements We wish to acknowledge the valuable assistance of the public veterinary practitioners in the region of Macedonia. We are grateful to the farmers who readily allowed us to bleed their herds. Our thanks are also due to F.F.J. Franssen and M. de Groot for technical assistance in serology. Finally, we are indebted to Prof. G. Uilenberg for reviewing the manuscript. This work was partially financed by Diagnostic Parasitaire (Institut de Zoologie), Neuch,~tei, Switzerland.
References Adam, K.M.G. and Blewett, D.A., 1978. A serological survey for Babesia in cattle in Scotland. II. The occurenceof antibodyin the populationand the distributionof infectedherds. Ann. Trop.Med. Parasit., 72 (5): 417-428.
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