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Seroprevalence of Babesia ovis in sheep in Catalonia, northeastern Spain
Veterinary Parasitology 79 (1998) 275±281
Seroprevalence of Babesia ovis in sheep in Catalonia, northeastern Spain David Ferrer*, Joaquim CastellaÁ, J.F. GutieÂrrez Unitat de Parasitologia i Malalties ParasitaÁries, Facultat de VeterinaÁria, Universitat AutoÁnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain Received 2 February 1998; accepted 28 April 1998
Abstract An indirect fluorescent antibody test (IFAT) was developed in order to detect antibodies against Babesia ovis in different sheep flocks from Catalonia, northeastern Spain. Of the total 2174 sera included in the study, 133 (6.1%) were positive. Considerable differences in seroprevalence were detected depending on the geographical areas from which the animals originated. This must have been a consequence of the presence or absence of the vector tick, Rhipicephalus bursa, the distribution of which depends upon the bioclimate and vegetation present in the different areas studied. Even within those areas which afford suitable conditions for the presence of Rh. bursa ticks, and therefore in which positive titres were found, great differences in seroprevalence were detected among different flocks. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Babesia ovis; Sheep; Serology; Epidemiology-protozoa; IFAT; Spain
1. Introduction Babesia are intraerythrocytic protozoan parasites of domestic and wild animals and are a cause of anaemia and haemoglobinuria. Babesiosis is among the tick-borne diseases that cause the greatest economic losses in Spanish sheep production (Habela et al., 1990b). It can also affect goats, though more rarely (Yeruham et al., 1992). Babesiosis in small ruminants is due to, at least, three parasite species, namely, B. ovis, B. motasi and B. crassa. The taxonomy of the Babesia spp. of sheep and goats is not well-defined, however (Friedhoff, 1988). Among them, B. ovis is the most pathogenic. Sheep babesiosis * Corresponding author. Tel.: +3581 10 49; fax: +3581 20 06; e-mail: [email protected] 0304-4017/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 7 5 - 7
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caused by B. ovis is known to occur in the Mediterranean basin as well as in other areas where the vector tick is present (Yeruham et al., 1992). B. ovis is transmitted by the twohost tick Rhipicephalus bursa. This tick species is widespread between the latitudes 318 and 458N (Yeruham et al., 1985). The only published work on the seroprevalence of this ovine piroplasm in Spain is that of Habela et al. (1990b) in the southwestern part of the country. The aim of this work was to determine the seroprevalence of B. ovis in sheep in Catalonia, northeastern Spain, and to compare it in areas with different bioclimatic and vegetational conditions, and to study the influence of different territories of pasture. For this purpose, an indirect fluorescent antibody test (IFAT) was developed using a B. ovis isolate of ovine origin. 2. Materials and methods 2.1. Antigen preparation Blood was collected from several animals suffering from acute babesiosis in a sheep flock in western Spain. Blood samples were scanned for the presence of B. ovis, positive samples mixed and inoculated intravenously into a previously splenectomized sheep. Rectal temperature was recorded daily. The parasitaemia was also monitored daily by examination of thin blood films stained with Giemsa. Blood for IFAT antigen preparation was drawn at maximum parasitaemia of 2%, which occurred at 5 days post-infection. Venous blood was drawn and diluted 1:20 with phosphate-buffered saline (PBS), pH 7.2 immediately after bleeding and washed four times at 700 g for 10 min with PBS. The final sediment of washed erythrocytes was restored to the initial blood volume. This final suspension was dispensed in drop preparations on 10-well, glass slides, using a special applicator (Christensson, 1986). Once allowed to dry, they were fixed in ice-cold acetone, wrapped in paper towelling and sealed in plastic bags with silica gel to protect them from excess humidity. Slides were kept at ÿ808C until used. 2.2. Field study area and `sera samples The area studied comprises 1500 km2 and is situated between 408300 ±418150 N and 08100 ±08550 E. It was divided into three zones according to bioclimatic and vegetational factors. The first one comprises the river Ebre delta and the surrounding irrigated crops, mainly rice, and other cultivated lands with different vegetables and fruit trees. This first zone is situated between sea level and 150 m and has a coastal Mediterranean climate. The second zone consists mainly of typically Mediterranean scrubland of sclerophyllous shrubs (e.g. Quercus coccifera) generating a dense undergrowth as well as arboreal vegetation represented primarily by Quercus ilex and several pine species, specially Pinus halepensis. The only cultivated lands are dedicated to olive, almond and carob trees. This zone is situated below 600 m and presents an attenuated Mediterranean bioclimate. In the third zone, which is situated between 600 and 1100 m, a mountainous submediterranean bioclimate predominates, with no agriculture, and arboreal vegetation mainly consisting of deciduous (oak and beech) and coniferous (Pinus silvestris and Pinus nigra salzmanii)
D. Ferrer et al. / Veterinary Parasitology 79 (1998) 275±281
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Table 1 Number of flocks and sheep included in the study
Number of flocks Number of sera
Zone 1
Zone 2
Zone 3
Total
14 (28%) 385 (17.7%)
3 (6%) 368 (16.9%)
33 (66%) 1421 (65.4)%
50 (100%) 2174 (100%)
forests. These are highland pastures that domestic ruminants share during the warm season with mouflon (Ovis musimon) and a large population of Spanish ibex (Capra pyrenaica). Sera were collected from 1995 to 1997 from 2174 domestic sheep belonging to 50 flocks in southern Catalonia, Spain. The mean number of sheep per flock was 86 (range: 10±300). Twenty-six of the 50 farms included in the study reared sheep and goats together, while the other 24 exclusively reared sheep. The number of sheep and flocks in each of the three zones that were included in the study are shown in Table 1. The number of sera studied in each flock in order to determine the seroprevalence was determined using the WinEpiscope software. An expected prevalence of 5% was chosen, as well as a confidence level of 95% and an accepted error of 5%. The expected prevalence was modified in the course of the study for those flocks showing an actual prevalence higher than 5%. The mean number of sera studied per flock was 43 (range: 2±114). Sera were stored at ÿ308C until used. Positive and negative control sera were kindly provided by Dr. M.A. Habela, who had already carried out an IFAT survey on the seroprevalence of B. ovis in southwestern Spain (Habela et al., 1990b). This positive control showed an IFAT titre of 1:640. 2.3. IFAT procedure Antigen slides were incubated overnight with 1% bovine serum albumin in PBS (pH 7.2) to avoid nonspecific antibody binding in the subsequent incubations. Slides were washed three times for 5 min each with 0.05% Tween-20 in PBS (PBS-T). Test sera were used at dilutions ranging from 1:160 to 1:2560 for titration and at 1:160 for the determination of infection prevalence. This latter titre was selected because it had given the best sensitivity and specificity results in a previous checkerboard titration with positive and negative control sera. Moreover, it had already been used in other IFAT surveys in Spain (Habela et al., 1990b). Serum drops were dispensed into each slide well. The slides were subsequently incubated at 378C in a humid chamber for 30 min and then washed three times in PBS-T. Commercial fluorescein isothiocyanate-conjugated rabbit anti-sheep IgG (Sigma, St. Louis, USA) was used at a dilution of 1:160 in PBS. The slides were incubated and washed as before. They were dried, mounted in buffered glycerine (9 parts of glycerine and 1 part of PBS) and examined immediately with an Olympus fluorescence microscope at a magnification of 400. Definitive titres were determined as the lowest dilution of sera giving a positive fluorescence pattern, consisting of one or two fluorescent spots inside the infected erythrocytes, which corresponded to the Babesia merozoites.
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Table 2 Babesia ovis titres in sheep Zone
Titres <1:160
1:160
1:320
1:640
1:1280
1:2560
Zone 1
383
1
0
1
0
0
Zone 2
348
7
4
3
6
0
Zone 3
1310
30
31
28
21
1
Total (n2174)
2041
38 (28.6%)
35 (26.3%)
32 (24%)
27 (20.3%)
1 (0.75%)
Positive animals 2/385 (0.52%) 20/368 (5.43%) 111/1421 (7.8%) 133/2174 (6.1%)
Positive flocks 1/14 (7.14%) 2/3 (66.6%) 22/33 (66.6%) 25/50 (50%)
3. Results Of the 2174 sera tested, 133 (6.1%) showed titres ranging from 1:160 to 1:2560 and were thus considered positive. The overall results of the IFAT as well as those recorded specifically in each of the three zones are shown in Table 2. Only two positive results were recorded in one flock from the first zone, while the positivity rates for the second and third zones were 5.43 and 7.8%, respectively. The prevalences found in each flock are depicted in Fig. 1. The highest prevalence was found in one flock from the third zone, which showed a 62.9% of positive titres (39 out of 62 animals studied). Prevalences higher than 10% were found in seven flocks, all of which belonged to zone 3.
Fig. 1. Prevalence of B. ovis in flocks.
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4. Discussion The overall seroprevalence found in sheep in the studied area was 6.1%, considerably lower than those reported by other researchers. Habela et al. (1990b) found 57.7% positive titres in 97 sheep from Extremadura, southern Spain, using the same specificity level of 1:160. Yeruham et al. (1995) found a positive serological response in 84.5% of hoggets (442 of 523) and 88.9% of ewes (921 of 1036) in Israel. In Macedonia, Papadopoulos et al. (1996b) reported a seroprevalence of 52.1% in sheep (375 of 720) and 36.4% in goats (177 of 486). All these values, however, were obtained in enzootic areas of sheep babesiosis, while in our study, samples were obtained from a heterogeneous area with very different bioclimatic conditions. Serological cross-reactivity between species of Babesia in small ruminants was studied by Papadopoulos et al. (1996a) in Greece. These authors reported that cross-reactions between Theileria ovis and the three Babesia spp. of small ruminants are non-existing or weak. They also concluded that there are common epitopes between B. motasi and B. crassa, and to some extent also between them and B. ovis. According to these crossreactions, found only at low dilutions, they fixed the specificity level of the test at 1:160 in order to avoid false-positive results. This cut-off titre is exactly the same we fixed for our IFAT. In another study carried out in Spain, Habela et al. (1990b) found no cross-reaction between B. ovis antigens and B. motasi, B. crassa, or Th. ovis. The levels of positivity to B. ovis have been extremely variable between and within some of the three zones in which the studied area was divided. The distribution of positive titres, and thus of B. ovis, is based upon the existence of Rhipicephalus bursa, the only known vector for this piroplasm, which directly depends upon the bioclimatic and vegetational conditions. Rh. bursa is a two-host tick which requires savannah-like sclerophyllous vegetation, especially when it consists of herbaceous and bushy plants, as well as Quercus pubescens, Pinus pinea and Olea europaea in the arboreous stratum (Travassos, 1994). This tick is rarely found in the first zone, mainly because it lacks the necessary undergrowth for its survival. That is why the seroprevalence there was only 0.52%. We have not been able to find a definite explanation for the two sheep positive in this zone, both belonging to the same flock. If they had been purchased from a flock in a positive zone before the sampling, however, their titres would have persisted for several months despite being no longer in contact with B. ovis (Habela et al., 1990a). The most common tick found on sheep in zones 1 and 2 is Rhipicephalus turanicus. The role of this tick in the transmission of B. ovis is not well determined, although the extremely low seroprevalence in zone 1 makes us think that it does not act as a vector of this piroplasm. Rhipicephalus bursa is most frequent in zone 3. Both Rh. bursa and Rh. turanicus are found during the warm season, when cases of ovine babesiosis are reported. In zone 3 Dermacentor marginatus can also be found, but only in the cold season. Flocks from localities in zone 1 always graze pastures in their same zone, that is, the lowlands, while some flocks from zone 2 regularly move to higher pastures located in zone 3, specially in spring and summer. Almost all positive titres were found in zones 2 and 3. The rate of positive animals was higher in zone 3 (7.8% vs. 5.4%). As has been mentioned before, however, the
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distribution of positive results has been extremely irregular within these two regions. Positivity rates should logically depend on the probability of being in contact with B. ovis, and consequently with infected Rh. bursa ticks. The main factor affecting this probability is the pastures which are grazed by each of the flocks included in these two zones. Thus, if different flocks from the same locality graze very distant pastures, each showing different phytoclimatic conditions, then the respective prevalences should be expected to differ considerably. That is why, for example, the positivity rates in two different flocks from one locality in zone 3 were 4.7 (2 of 42) and 62.9% (39 of 62). We think that there might be another factor playing an important role in the transmission of ovine babesiosis in grazing sheep, especially in those moving to zone 3. Here domestic ruminants occasionally share pastures with a large population of Spanish ibex (Capra pyrenaica) in the warm season. Adult Rh. bursa activity lasts from March to September, though it is most intense from May to June, while larvae and nymphs are more frequently found during autumn and winter (Travassos, 1994). We have repeatedly observed that domestic ruminants moving to the high pastures in the warm season are infested by large numbers of adult ticks whose immature stages have fed on Spanish ibex during the winter. B. ovis is transmitted transstadially and transovarially in Rh. bursa. Thus, this wild ruminant would be a major factor responsible for the maintenance of the vector tick population and consequently for the maintenance of B. ovis during the cold season. Recently, Ferrer et al. (1998) reported a 32.6% seroprevalence of B. ovis by IFAT in 475 Spanish ibex from the same geographical area where our present study was carried out. In addition to this, sporokinetes of Babesia spp. have been documented in the haemolymph of female Rhipicephalus bursa ticks that had fed on these wild ruminants (CastellaÁ et al., 1995). These authors were not able, however, to conclude whether these sporokinetes in the arthropod vector belonged to B. ovis or to a different Babesia species. Acknowledgements We thank Andreu Gil and Delmir Prieto for their invaluable help. References CastellaÁ, J., Ferrer, D., Estrada, A., 1995. MorfometrõÂa de los esporoquinetos de Babesia sp. hallados en hemolinfas de Rhipicephalus bursa. III Simposium IbeÂrico sobre Ixodoidea y Enfermedades Transmitidas, Alcala de Henares, Spain. Christensson, D., 1986. Improvement of the teflonized slides used in the immunofluorescent antibody technique. Acta Vet. Scand. 27, 296±298. Ferrer, D., CastellaÁ, J., GutieÂrrez, J.F., LavõÂn, S., Marco, I., 1998. Seroprevalence of Babesia ovis in Spanish ibex (Capra pyrenaica) in Catalonia, northeastern Spain. Vet. Parasitol. 75, 91±96. Friedhoff, K.T., 1988. Transmission of Babesia. In: Ristic, M. (Ed.), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, FL, 36 pp. Habela, M., Reina, D., Nieto, C., Navarrete, I., 1990a. Antibody response and duration of latent infection in sheep following experimental infection with Babesia ovis, Vet. Parasitol. 35, pp. 1±10.
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Habela, M., Reina, D., Nieto, C., Navarrete, I., 1990b. Isolation and identification of Babesia ovis in Extremadura (Spain). Vet. Parasitol. 35, pp. 233±238. Papadopoulos, B., PerieÂ, N.M., Uilenberg, G., 1996a. Piroplasms of domestic animals in the Macedonia region of Greece. 1. Serological cross-reactions. Vet. Parasitol. 63, pp. 41±56. Papadopoulos, B., Brossard, M., PerieÂ, N.M., 1996b. Piroplasms of domestic animals in the Macedonia region of Greece. 3. Piroplasms of small ruminants. Vet. Parasitol. 63, pp. 67±74. Travassos, J.A., 1994. As carrac,as (Acarina:Ixodoidea) da PenõÂnsula IbeÂrica. In: Estudos, Ensaios e Documentos no. 158, Instituto de Investigac,aÄo CientõÂfica Tropical, Lisboa, 163 pp. Yeruham, I., Hadani, A., Galker, F., Mauer, E., Rubina, M., Rosen, S., 1985. The geographical distribution and animal hosts of Rhipicephalus bursa (Canestrini and Fanzago, 1877) in Israel. Rev. Elev. MeÂd. VeÂt. Pays Trop. 38, 173±179. Yeruham, I., Hadani, A., Galker, F., Rosen, S., Schlien, J., 1992. A field study of haemoparasites in two flocks of sheep in Israel. Isr. J. Vet. Med. 47, 107±111. Yeruham, I., Hadani, A., Galker, F., Rosen, S., 1995. A study of an enzootic focus of sheep babesiosis (Babesia ovis, Babes, 1892). Vet. Parasitol. 60, 349±354.
Seroprevalence of Babesia ovis in sheep in Catalonia, northeastern Spain David Ferrer*, Joaquim CastellaÁ, J.F. GutieÂrrez Unitat de Parasitologia i Malalties ParasitaÁries, Facultat de VeterinaÁria, Universitat AutoÁnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain Received 2 February 1998; accepted 28 April 1998
Abstract An indirect fluorescent antibody test (IFAT) was developed in order to detect antibodies against Babesia ovis in different sheep flocks from Catalonia, northeastern Spain. Of the total 2174 sera included in the study, 133 (6.1%) were positive. Considerable differences in seroprevalence were detected depending on the geographical areas from which the animals originated. This must have been a consequence of the presence or absence of the vector tick, Rhipicephalus bursa, the distribution of which depends upon the bioclimate and vegetation present in the different areas studied. Even within those areas which afford suitable conditions for the presence of Rh. bursa ticks, and therefore in which positive titres were found, great differences in seroprevalence were detected among different flocks. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Babesia ovis; Sheep; Serology; Epidemiology-protozoa; IFAT; Spain
1. Introduction Babesia are intraerythrocytic protozoan parasites of domestic and wild animals and are a cause of anaemia and haemoglobinuria. Babesiosis is among the tick-borne diseases that cause the greatest economic losses in Spanish sheep production (Habela et al., 1990b). It can also affect goats, though more rarely (Yeruham et al., 1992). Babesiosis in small ruminants is due to, at least, three parasite species, namely, B. ovis, B. motasi and B. crassa. The taxonomy of the Babesia spp. of sheep and goats is not well-defined, however (Friedhoff, 1988). Among them, B. ovis is the most pathogenic. Sheep babesiosis * Corresponding author. Tel.: +3581 10 49; fax: +3581 20 06; e-mail: [email protected] 0304-4017/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 7 5 - 7
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caused by B. ovis is known to occur in the Mediterranean basin as well as in other areas where the vector tick is present (Yeruham et al., 1992). B. ovis is transmitted by the twohost tick Rhipicephalus bursa. This tick species is widespread between the latitudes 318 and 458N (Yeruham et al., 1985). The only published work on the seroprevalence of this ovine piroplasm in Spain is that of Habela et al. (1990b) in the southwestern part of the country. The aim of this work was to determine the seroprevalence of B. ovis in sheep in Catalonia, northeastern Spain, and to compare it in areas with different bioclimatic and vegetational conditions, and to study the influence of different territories of pasture. For this purpose, an indirect fluorescent antibody test (IFAT) was developed using a B. ovis isolate of ovine origin. 2. Materials and methods 2.1. Antigen preparation Blood was collected from several animals suffering from acute babesiosis in a sheep flock in western Spain. Blood samples were scanned for the presence of B. ovis, positive samples mixed and inoculated intravenously into a previously splenectomized sheep. Rectal temperature was recorded daily. The parasitaemia was also monitored daily by examination of thin blood films stained with Giemsa. Blood for IFAT antigen preparation was drawn at maximum parasitaemia of 2%, which occurred at 5 days post-infection. Venous blood was drawn and diluted 1:20 with phosphate-buffered saline (PBS), pH 7.2 immediately after bleeding and washed four times at 700 g for 10 min with PBS. The final sediment of washed erythrocytes was restored to the initial blood volume. This final suspension was dispensed in drop preparations on 10-well, glass slides, using a special applicator (Christensson, 1986). Once allowed to dry, they were fixed in ice-cold acetone, wrapped in paper towelling and sealed in plastic bags with silica gel to protect them from excess humidity. Slides were kept at ÿ808C until used. 2.2. Field study area and `sera samples The area studied comprises 1500 km2 and is situated between 408300 ±418150 N and 08100 ±08550 E. It was divided into three zones according to bioclimatic and vegetational factors. The first one comprises the river Ebre delta and the surrounding irrigated crops, mainly rice, and other cultivated lands with different vegetables and fruit trees. This first zone is situated between sea level and 150 m and has a coastal Mediterranean climate. The second zone consists mainly of typically Mediterranean scrubland of sclerophyllous shrubs (e.g. Quercus coccifera) generating a dense undergrowth as well as arboreal vegetation represented primarily by Quercus ilex and several pine species, specially Pinus halepensis. The only cultivated lands are dedicated to olive, almond and carob trees. This zone is situated below 600 m and presents an attenuated Mediterranean bioclimate. In the third zone, which is situated between 600 and 1100 m, a mountainous submediterranean bioclimate predominates, with no agriculture, and arboreal vegetation mainly consisting of deciduous (oak and beech) and coniferous (Pinus silvestris and Pinus nigra salzmanii)
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Table 1 Number of flocks and sheep included in the study
Number of flocks Number of sera
Zone 1
Zone 2
Zone 3
Total
14 (28%) 385 (17.7%)
3 (6%) 368 (16.9%)
33 (66%) 1421 (65.4)%
50 (100%) 2174 (100%)
forests. These are highland pastures that domestic ruminants share during the warm season with mouflon (Ovis musimon) and a large population of Spanish ibex (Capra pyrenaica). Sera were collected from 1995 to 1997 from 2174 domestic sheep belonging to 50 flocks in southern Catalonia, Spain. The mean number of sheep per flock was 86 (range: 10±300). Twenty-six of the 50 farms included in the study reared sheep and goats together, while the other 24 exclusively reared sheep. The number of sheep and flocks in each of the three zones that were included in the study are shown in Table 1. The number of sera studied in each flock in order to determine the seroprevalence was determined using the WinEpiscope software. An expected prevalence of 5% was chosen, as well as a confidence level of 95% and an accepted error of 5%. The expected prevalence was modified in the course of the study for those flocks showing an actual prevalence higher than 5%. The mean number of sera studied per flock was 43 (range: 2±114). Sera were stored at ÿ308C until used. Positive and negative control sera were kindly provided by Dr. M.A. Habela, who had already carried out an IFAT survey on the seroprevalence of B. ovis in southwestern Spain (Habela et al., 1990b). This positive control showed an IFAT titre of 1:640. 2.3. IFAT procedure Antigen slides were incubated overnight with 1% bovine serum albumin in PBS (pH 7.2) to avoid nonspecific antibody binding in the subsequent incubations. Slides were washed three times for 5 min each with 0.05% Tween-20 in PBS (PBS-T). Test sera were used at dilutions ranging from 1:160 to 1:2560 for titration and at 1:160 for the determination of infection prevalence. This latter titre was selected because it had given the best sensitivity and specificity results in a previous checkerboard titration with positive and negative control sera. Moreover, it had already been used in other IFAT surveys in Spain (Habela et al., 1990b). Serum drops were dispensed into each slide well. The slides were subsequently incubated at 378C in a humid chamber for 30 min and then washed three times in PBS-T. Commercial fluorescein isothiocyanate-conjugated rabbit anti-sheep IgG (Sigma, St. Louis, USA) was used at a dilution of 1:160 in PBS. The slides were incubated and washed as before. They were dried, mounted in buffered glycerine (9 parts of glycerine and 1 part of PBS) and examined immediately with an Olympus fluorescence microscope at a magnification of 400. Definitive titres were determined as the lowest dilution of sera giving a positive fluorescence pattern, consisting of one or two fluorescent spots inside the infected erythrocytes, which corresponded to the Babesia merozoites.
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Table 2 Babesia ovis titres in sheep Zone
Titres <1:160
1:160
1:320
1:640
1:1280
1:2560
Zone 1
383
1
0
1
0
0
Zone 2
348
7
4
3
6
0
Zone 3
1310
30
31
28
21
1
Total (n2174)
2041
38 (28.6%)
35 (26.3%)
32 (24%)
27 (20.3%)
1 (0.75%)
Positive animals 2/385 (0.52%) 20/368 (5.43%) 111/1421 (7.8%) 133/2174 (6.1%)
Positive flocks 1/14 (7.14%) 2/3 (66.6%) 22/33 (66.6%) 25/50 (50%)
3. Results Of the 2174 sera tested, 133 (6.1%) showed titres ranging from 1:160 to 1:2560 and were thus considered positive. The overall results of the IFAT as well as those recorded specifically in each of the three zones are shown in Table 2. Only two positive results were recorded in one flock from the first zone, while the positivity rates for the second and third zones were 5.43 and 7.8%, respectively. The prevalences found in each flock are depicted in Fig. 1. The highest prevalence was found in one flock from the third zone, which showed a 62.9% of positive titres (39 out of 62 animals studied). Prevalences higher than 10% were found in seven flocks, all of which belonged to zone 3.
Fig. 1. Prevalence of B. ovis in flocks.
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279
4. Discussion The overall seroprevalence found in sheep in the studied area was 6.1%, considerably lower than those reported by other researchers. Habela et al. (1990b) found 57.7% positive titres in 97 sheep from Extremadura, southern Spain, using the same specificity level of 1:160. Yeruham et al. (1995) found a positive serological response in 84.5% of hoggets (442 of 523) and 88.9% of ewes (921 of 1036) in Israel. In Macedonia, Papadopoulos et al. (1996b) reported a seroprevalence of 52.1% in sheep (375 of 720) and 36.4% in goats (177 of 486). All these values, however, were obtained in enzootic areas of sheep babesiosis, while in our study, samples were obtained from a heterogeneous area with very different bioclimatic conditions. Serological cross-reactivity between species of Babesia in small ruminants was studied by Papadopoulos et al. (1996a) in Greece. These authors reported that cross-reactions between Theileria ovis and the three Babesia spp. of small ruminants are non-existing or weak. They also concluded that there are common epitopes between B. motasi and B. crassa, and to some extent also between them and B. ovis. According to these crossreactions, found only at low dilutions, they fixed the specificity level of the test at 1:160 in order to avoid false-positive results. This cut-off titre is exactly the same we fixed for our IFAT. In another study carried out in Spain, Habela et al. (1990b) found no cross-reaction between B. ovis antigens and B. motasi, B. crassa, or Th. ovis. The levels of positivity to B. ovis have been extremely variable between and within some of the three zones in which the studied area was divided. The distribution of positive titres, and thus of B. ovis, is based upon the existence of Rhipicephalus bursa, the only known vector for this piroplasm, which directly depends upon the bioclimatic and vegetational conditions. Rh. bursa is a two-host tick which requires savannah-like sclerophyllous vegetation, especially when it consists of herbaceous and bushy plants, as well as Quercus pubescens, Pinus pinea and Olea europaea in the arboreous stratum (Travassos, 1994). This tick is rarely found in the first zone, mainly because it lacks the necessary undergrowth for its survival. That is why the seroprevalence there was only 0.52%. We have not been able to find a definite explanation for the two sheep positive in this zone, both belonging to the same flock. If they had been purchased from a flock in a positive zone before the sampling, however, their titres would have persisted for several months despite being no longer in contact with B. ovis (Habela et al., 1990a). The most common tick found on sheep in zones 1 and 2 is Rhipicephalus turanicus. The role of this tick in the transmission of B. ovis is not well determined, although the extremely low seroprevalence in zone 1 makes us think that it does not act as a vector of this piroplasm. Rhipicephalus bursa is most frequent in zone 3. Both Rh. bursa and Rh. turanicus are found during the warm season, when cases of ovine babesiosis are reported. In zone 3 Dermacentor marginatus can also be found, but only in the cold season. Flocks from localities in zone 1 always graze pastures in their same zone, that is, the lowlands, while some flocks from zone 2 regularly move to higher pastures located in zone 3, specially in spring and summer. Almost all positive titres were found in zones 2 and 3. The rate of positive animals was higher in zone 3 (7.8% vs. 5.4%). As has been mentioned before, however, the
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distribution of positive results has been extremely irregular within these two regions. Positivity rates should logically depend on the probability of being in contact with B. ovis, and consequently with infected Rh. bursa ticks. The main factor affecting this probability is the pastures which are grazed by each of the flocks included in these two zones. Thus, if different flocks from the same locality graze very distant pastures, each showing different phytoclimatic conditions, then the respective prevalences should be expected to differ considerably. That is why, for example, the positivity rates in two different flocks from one locality in zone 3 were 4.7 (2 of 42) and 62.9% (39 of 62). We think that there might be another factor playing an important role in the transmission of ovine babesiosis in grazing sheep, especially in those moving to zone 3. Here domestic ruminants occasionally share pastures with a large population of Spanish ibex (Capra pyrenaica) in the warm season. Adult Rh. bursa activity lasts from March to September, though it is most intense from May to June, while larvae and nymphs are more frequently found during autumn and winter (Travassos, 1994). We have repeatedly observed that domestic ruminants moving to the high pastures in the warm season are infested by large numbers of adult ticks whose immature stages have fed on Spanish ibex during the winter. B. ovis is transmitted transstadially and transovarially in Rh. bursa. Thus, this wild ruminant would be a major factor responsible for the maintenance of the vector tick population and consequently for the maintenance of B. ovis during the cold season. Recently, Ferrer et al. (1998) reported a 32.6% seroprevalence of B. ovis by IFAT in 475 Spanish ibex from the same geographical area where our present study was carried out. In addition to this, sporokinetes of Babesia spp. have been documented in the haemolymph of female Rhipicephalus bursa ticks that had fed on these wild ruminants (CastellaÁ et al., 1995). These authors were not able, however, to conclude whether these sporokinetes in the arthropod vector belonged to B. ovis or to a different Babesia species. Acknowledgements We thank Andreu Gil and Delmir Prieto for their invaluable help. References CastellaÁ, J., Ferrer, D., Estrada, A., 1995. MorfometrõÂa de los esporoquinetos de Babesia sp. hallados en hemolinfas de Rhipicephalus bursa. III Simposium IbeÂrico sobre Ixodoidea y Enfermedades Transmitidas, Alcala de Henares, Spain. Christensson, D., 1986. Improvement of the teflonized slides used in the immunofluorescent antibody technique. Acta Vet. Scand. 27, 296±298. Ferrer, D., CastellaÁ, J., GutieÂrrez, J.F., LavõÂn, S., Marco, I., 1998. Seroprevalence of Babesia ovis in Spanish ibex (Capra pyrenaica) in Catalonia, northeastern Spain. Vet. Parasitol. 75, 91±96. Friedhoff, K.T., 1988. Transmission of Babesia. In: Ristic, M. (Ed.), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, FL, 36 pp. Habela, M., Reina, D., Nieto, C., Navarrete, I., 1990a. Antibody response and duration of latent infection in sheep following experimental infection with Babesia ovis, Vet. Parasitol. 35, pp. 1±10.
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