Ž .Veterinary Parasitology 75 1998 93–98

Seroprevalence of Babesia oÕis in Spanish ibexž /Capra pyrenaica in Catalonia, northeastern Spain

David Ferrer a,), Joaquim Castella a, Juan F. Gutierrez a,` ´Santiago Lavın b, Ignasi Marco b´

a Unitat de Parasitologia i Malalties Parasitaries, Facultat de Veterinaria, UniÕersitat Autonoma de` ` `Barcelona, 08193 Bellaterra, Barcelona, Spain

b Unitat de Patologia General i Medica, Facultat de Veterinaria, UniÕersitat Autonoma de Barcelona,` ` `08193 Bellaterra, Barcelona, Spain

Received 28 July 1997; accepted 27 September 1997

Abstract

A serologic survey was carried out in order to detect antibodies against Babesia oÕis in a largeŽ .population of Spanish ibex Capra pyrenaica from a hunting reserve in Catalonia, northeastern

Ž .Spain. For this purpose, an indirect fluorescent antibody test IFAT was developed using a B.Ž .oÕis isolate of ovine origin as antigen. Of the total 475 sera tested, 155 32.6% showed titres

between 1:160 and 1:1280 and were considered positive. These results reveal that exposure ofSpanish ibex to B. oÕis is common in the studied area. No significant differences could bedetected when comparing season or year of capture and age or sex of the animals in positive andnegative samples. A high proportion of low titres was found in comparison to those reported byother researchers in sheep in Spain; this could be a consequence of the existence of some minorantigenic differences between B. oÕis of domestic sheep and that found in Spanish ibex. q 1998Elsevier Science B.V.

Keywords: Babesia oÕis; Ibex; Capra pyrenaica; Serology; Epidemiology—protozoa; IFAT; Spain

1. Introduction

Babesia are intraerythrocytic protozoan parasites of domestic and wild animals andare a cause of anaemia and haemoglobinuria. Babesia spp. have been documented inseveral wild ruminant species. In the United States Babesia odocoilei has been isolated

) Corresponding author. Tel.: q34-3-581-10-49; fax: q34-3-581-20-06.

0304-4017r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0304-4017 97 00199-4

( )D. Ferrer et al.rVeterinary Parasitology 75 1998 93–9894

Ž . Ž .from white-tailed deer Odocoileus Õirginianus in Texas Emerson and Wright, 1968Ž .and Oklahoma Waldrup et al., 1989 . In that country other Babesia spp. have been

Ž . Ž .isolated from bighorn sheep OÕis canadensis in California Goff et al., 1993 ;Ž . Ž . ŽThomford et al., 1993 , mule deer Odocoileus hemionus also in California Thomford

. Ž . Ž .et al., 1993 , caribou Rangifer tarandus caribou in Minnesota Holman et al., 1994aŽ . Ž .and North American elk CerÕus elaphus in Texas Holman et al., 1994b . In Europe

some reported findings of Babesia capreoli have been made since its first description byŽ . Ž .Enigk and Friedhoff 1962 in roe deer Capreolus capreolus . B. capreoli has also

Ž . Ž .been documented in red deer C. elaphus in Scotland Blewett and Adam, 1978 andŽ . Ž . Ž .sika deer CerÕus nippon in Ireland Gray et al., 1991 . Hinaidy 1987 reported latent

asymptomatic Babesia infections in red deer and roe deer in Austria as well as acuteŽ .fatal cases of babesiosis in Pere David’s deer Elaphurus daÕidianus . Finally, this`

Ž .protozoan has also been reported in sika deer from Japan Takahashi et al., 1992 .Babesia spp., however, has not been reported previously in wild ruminants in Spainthough there is evidence of the existence of some Babesia spp. in Spanish ibex in thearea where our study was carried out. Clinical babesiosis due to Babesia oÕis isregularly reported in domestic sheep and goats that share pastures with this Spanish ibexpopulation. Babesiosis in domestic small ruminants is due to, at least, three parasitespecies, namely B. oÕis, Babesia motasi and Babesia crassa. Among them, B. oÕis isthe most pathogenic. Sheep babesiosis caused by B. oÕis is known to occur in the

ŽMediterranean basin as well as in other areas where the vector tick is present Yeruham.et al., 1992 . B. oÕis is transmitted by the two-host tick Rhipicephalus bursa. This tick

Ž .species is widespread between the latitudes 318 and 458 N Yeruham et al., 1985 .Babesiosis is among the tick-borne diseases that cause the greatest economic losses in

Ž .Spanish sheep production Habela et al., 1990 . It can also affect goats, though moreŽ .rarely Yeruham et al., 1992 . The aim of this work was to determine the seroprevalence

Ž .of B. oÕis in a large population of Spanish ibex Capra pyrenaica in the NationalHunting Reserve of the Ports de Tortosa mountains in Catalonia, northeastern Spain. For

Ž .this purpose, an indirect fluorescent antibody test IFAT was developed using a B. oÕisisolate of ovine origin.

2. Materials and methods

2.1. Antigen preparation

Blood was collected from several animals suffering from acute babesiosis in a sheepflock in western Spain. Blood samples were scanned for the presence of B. oÕis,positive samples mixed and inoculated intravenously into a previously splenectomizedsheep. Rectal temperature was recorded daily. The parasitaemia was also monitoreddaily by examination of thin blood films stained with Giemsa. Blood for IFAT antigenpreparation was drawn at maximum parasitaemia of 2%, which occurred at 5 dayspostinfection. Venous blood was drawn and diluted 1:20 with phosphate-buffered-salineŽ .PBS , pH 7.2 immediately after bleeding and washed four times at 700=g for 10 minwith PBS. The final sediment of washed erythrocytes was restored to the initial blood

( )D. Ferrer et al.rVeterinary Parasitology 75 1998 93–98 95

volume. This final suspension was dispensed in drop preparations on 10-well, glassslides. Once allowed to dry, they were fixed in ice-cold acetone, wrapped in paper toweland sealed in plastic bags with silica gel to protect them from excess humidity. Slideswere kept at y808C until used.

2.2. Sera samples

Sera were collected by the Catalonia Department of Natural Resources personnelfrom Spanish ibex populations in Catalonia, northeastern Spain, from 1992 to 1995 aspart of a population control program. All samples were collected in the Hunting Reserve

Ž X X . 2of the Ports de Tortosa mountains 40849 N, 0820 E , which has an area of 301.5 km .In the beginning of the study, the population of this wild ruminant in the reserve wasestimated at 6291 animals. It represents the largest population of Spanish ibex in Spain.At the time of collection, approximate age, as well as sex and weight were recorded. Atotal of 475 serum samples was included in this study. Table 1 shows the age and sex of422 of the total 475 animals studied. In the remaining 53 animals these data could not berecorded.

ŽMost animals younger than 1 yr were captured when they were 5 or 6 months old 66.out of 77 or 85.7% . Sera were stored at y308C until used. Positive and negative control

sera were kindly provided by Dr. M.A. Habela, School of Veterinary Medicine, Caceres,´Spain.

2.3. IFAT procedure

ŽAntigen 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

Ž .washed three times for 5 min each with 0.05% Tween-20 in PBS PBS-T . Test serawere used at dilutions ranging from 1:160 to 1:2560 for titration and at 1:160 for thedetermination of infection prevalence. This latter titre was selected because it had

Ž .already been used in other surveys in Spain Habela et al., 1990 . Serum drops weredispensed into each slide well. The slides were subsequently incubated at 378C in ahumid chamber for 30 min and then washed three times in PBS-T. Commercial

Žfluorescein isothiocyanate-conjugated rabbit anti-goat IgG Sigma Chemical, St. Louis,.USA was used at a dilution of 1:160 in PBS. The slides were incubated and washed as

Table 1Age and sex of animals studied

Sex Age

-1 yr G1 yr Total

Ž . Ž .Y 40 14.3% 240 85.7% 280Ž . Ž .? 28 19.7% 114 80.3% 142

aTotal 68 354 422

aAge andror sex of 53 animals were not available.Total sera studied: 475.

( )D. Ferrer et al.rVeterinary Parasitology 75 1998 93–9896

Table 2B. oÕis titres in Spanish ibex

Sex Titres

-1:160 1:160 1:320 1:640 1:1280

Ž . Ž . Ž . Ž . Ž . Ž .Y ns280 184 65.7% 43 15.4% 42 15% 7 2.5% 4 1.4%Ž . Ž . Ž . Ž . Ž .? ns142 99 69.7% 22 15.5% 12 8.5% 9 6.3%

a Ž . Ž . Ž . Ž . Ž .U ns53 37 69.8% 6 11.3% 7 13.2% 3 5.7%Ž . Ž . Ž . Ž . Ž . Ž .Total ns475 320 67.4% 71 14.9% 61 12.8% 19 4% 4 0.8%

aU: sex not known.

Žbefore. They were dried, mounted in buffered glycerine nine parts of glycerine and one.part of PBS and examined immediately with an Olympus fluorescence microscope at a

magnification of =400. The definitive titres were determined as the lowest dilution ofsera giving a positive fluorescence pattern.

2.4. Statistical analysis

Statistical analysis of data obtained was performed using the SPSS software package.A confidence interval of 95% was used in the x 2 test of association to determinesignificance of the relationships between positive IFAT and the parameters of animalage, sex, and season and year of capture.

3. Results

Ž .Of the 475 sera tested, 155 32.6% showed titres ranging from 1:160 to 1:1280 andwere, thus, considered positive. The overall results of the IFAT are shown in Table 2. Ofthe known females and males tested, 34.3 and 30.2% yielded a positive result,respectively. In Fig. 1 the distribution of frequency by age is shown for animals older

Ž .than 1 yr. Out of 77 animals younger than 1 yr, 31 were positive 40.3% .

Fig. 1. Distribution of frequencies by ages for animals older than 1 yr.

( )D. Ferrer et al.rVeterinary Parasitology 75 1998 93–98 97

No statistically significant differences could be established between season and yearof capture or age and sex of the animals in positive and negative samples. Nobackground reactions were recorded with negative control sera at dilutions higher than1:160. This nonspecific background reaction was found indeed at lower dilutions whenthe checkerboard titration was made with negative control sera, specially at 1:20, but itcould be easily distinguished from the positive pattern of fluorescence for B. oÕis. Withpositive sera, the shape of infected erythrocytes as well as the presence of the protozoanwere clearly defined by the fluorescence distribution.

4. Discussion

Our results of seroprevalence show that exposure of Spanish ibex to B. oÕis iscommon in the studied area. No confirmed clinical cases of babesiosis have been

Ž .recorded in this population, though they are suspected unpublished data . Clinical casesof babesiosis due to B. oÕis, however, are regularly found in sheep which share pastureswith this wild ruminant. Moreover, B. oÕis has been recorded in blood smears from

Ž . Žmouflon OÕis musimon , another wild ruminant from the reserve personal communica-.tion .

Sporokinetes of Babesia spp. have been documented in the haemolymphs of femaleŽ .R. bursa ticks that had fed on these wild ruminants Castella et al., 1995 . These authors`

were not able, however, to conclude whether these sporokinetes in the arthropod vectorbelonged to B. oÕis or to a different Babesia species. R. bursa is the most common tickfound on both domestic and wild ruminants in this area, but neither Ixodes scapularisnor Ixodes ricinus, the respective vectors for B. odocoilei and B. capreoli, have beenrecorded.

Serological cross-reactivity between species of Babesia in small ruminants wasŽ .studied by Papadopoulos et al. 1996 in Greece. These authors concluded that there are

common epitopes between B. motasi and B. crassa, and to some extent also betweenŽ .them and B. oÕis. In Spain, Habela et al. 1990 found no cross-reaction between B.

oÕis antigens and B. motasi, B. crassa, or Theileria oÕis.Titres found in Spanish ibex were usually low compared to those reported by Habela

Ž .et al. 1990 in sheep from Spain. In the present study, 85% of positive samples showeda titre of 1:160 or 1:320 and the maximum detected titre was 1:1280. Habela reportedtitres as high as 1:5120 with 55.3% of positives between 1:640 and 1:5120. Consideringthe little cross-reactivity reported by the aforementioned authors between B. oÕis andthe other ovine piroplasms, we think that this discrepancy in titres between Spanish ibexand sheep might be due to the existence of minor antigenic differences between B. oÕisof domestic sheep and that found in Spanish ibex.

No significant differences in positivity were found when comparing animals older oryounger than 1 year. Such differences would have been expected if the captures hadbeen homogenously distributed by age. In that case most young animals captured beforetheir first contact with infected R. bursa, which usually takes place in the warm season,would have been negative. Most captures, however, were made between October andDecember; at this time nearly all the young animals had probably had contact with B.oÕis.

( )D. Ferrer et al.rVeterinary Parasitology 75 1998 93–9898

This study is a part of a larger serologic survey on the prevalence of antibodiesagainst B. oÕis in domestic sheep and goats, and was designed to address the influenceof wild ruminant hosts in the distribution, spread and maintenance of this protozoan indomestic animals which share their territory.

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