Serologic Survey of the Wild Boar (Sus scrofa) for Borrelia Burgdorferi ...

VECTOR-BORNE AND ZOONOTIC DISEASES Volume 9, Number 5, 2009 © Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2008.0125

ORIGINAL ARTICLE

Serologic Survey of the Wild Boar (Sus scrofa) for Borrelia Burgdorferi Sensu Lato Z. Jur˘ icová and Z. Hubálek

Abstract

Sera of 642 wild boars (Sus scrofa) shot by hunters in ten administrative regions of the Czech Republic during 1995–2000, were tested by indirect hemagglutination assay (IHA) for the presence of anti-Borrelia IgG. Antibodies to Borrelia burgdorferi sensu lato (Bb) were detected in serum samples from all 10 regions, and overall seroprevalence rate was 12.8%. Titres of antibodies ranged from 1:80 to 1:640. Borrelia antibodies were most frequent in the animals from three administrative regions of the Czech Republic: Moravskoslezsky´ (25.0%), Pardubicky´ (25.0%) and Královehradecky´ (24.1%), followed by the regions Plzenˇsky´ (16.7%), Olomoucky´ (13.3%), Jihomoravsky´ (12.8%), Vysocˇ ina (11.1%), Jihocˇ esky´ (11.1%), Zlínsky´ (10.3%), and Liberecky´ (8.9%). Seasonal seroprevalence rate increased in March and April, the peak was in May. The results suggest frequent exposure of wild boars to ixodid ticks infected with Bb, predominantly in rural and forested regions. The study also reviews the importance of wild boar in Lyme borreliosis (LB) ecology. Wild boar serology may provide another means of surveillance of endemic areas of LB. Key Words: Borrelia burgdorferi—Sus scrofa—serosurvey—Czech Republic.

Introduction

Materials and Methods

L

Blood sampling

yme borreliosis (LB) is a widespread zoonosis caused by infection with the tick-borne spirochete B. burgdorferi s. l. (Bb). Principal vector of LB in Central Europe is Ixodes ricinus (Piesman and Gern 2004). Natural habitats of this tick species are deciduous woodland and mixed forests. The ticks complete life cycle by obtaining blood meals from a wide range of vertebrate hosts. Not all tick-hosts are competent reservoirs of Bb. Some of them only serve as the source of blood for ticks but do not infect them with Bb: e.g., roe deer (Capreolus capreolus), red deer (Cervus elaphus), and fallow deer (Dama dama) (Jaenson and Tälleklint 1992, 1994; Gray et al. 1995; 1992; Piesman and Gern 2004). Wild boar can be included as an occasional host of Bb, as indicated previously by detection of antibodies to Bb in many endemic areas of Europe: France (Doby et al. 1991; Perez-Eid 1995), Bulgaria (Angelov et al. 1995), and two parts of the Czech Republic— south Moravia (Jur˘ icová et al. 1996, 2000), and central Bohemia (Zeman and Janus˘ka 1999). This study reports the results of a serological survey for antibodies to Bb in wild boars from the majority of regions of the Czech Republic (10 out of 14, see Fig. 1). Our aim was to determine whether the prevalence of antibodies to Bb in wild boars differs among regions of the Czech Republic.

Blood samples of hunter-killed wild boars were collected for serological analysis during 1995-1998 and in 2000. The sampling took place on the territory of ten administrative regions (and 37 districts) of the Czech Republic: Jihomoravsky´ (JM), Zlínsky´ (ZL), Moravskoslezsky´ (MS), Olomoucky´ (OC), Královehradecky´ (KH), Vysocˇina (VY), Jihocˇesky´ (JC), Pardubicky´ (PB), Liberecky´ (LB) and Plzen˘sky´ (PZ) (Fig. 1). Information on sites where wild boars were killed was provided by hunters to state veterinary officers at the checking stations. Blood specimens were collected with a syringe from the heart or thoracic cavity of 642 killed wild boars. The samples were centrifuged to separate sera which were then stored frozen at 20°C until analysis. Serology For demonstrating IgG antibodies to Bb, the indirect hemagglutination assay (IHA: BAG-Borrelia-HA-Test) was performed according to directions of the manufacturer (BAG-Biologische Analysensystem GmbH, Lich, Germany). Rehydrated tanned sheep erythrocytes fixed in glutaralde-

Institute of Vertebrate Biology, Academy of Sciences, Brno, Czech Republic

479

JURˇICOVÁ ET AL.

480 Table 1. Seroprevalence Rates in Wild Boars for Borrelia Burgdorferi S.L. in Regions Studied Region Jihomoravský (JM) Zlínský (ZL) Moravskoslezský (MS) Olomoucký (OC) Královehradecký Vysoˇc ina (VY) Jihoˇc eský (JC) Pardubický (PB) Liberecký (LB) Plzeˇnský (PZ) Total

Seroprevalence

%

26/203* 3/29 4/16 4/30 7/29 17/153 13/117 2/8 4/45 2/12 82/62

12.8 10.3 25.0 13.3 24.1 11.1 11.1 25.0 8.9 16.7 12.8

*Total positive/total examined.

hyde and bound with purified antigens from Bb strains (according to the manufacturer, antigens of B. burgdorferi sensu stricto, B. garinii, and B. afzelii have been pooled, i.e. those from all the three main human pathogenic genomic species in Europe, although differentiation among the species is impossible in this test) were used as test erythrocytes, while non-sensitized sheep erythrocytes fixed in glutaraldehyde as a control. Titres 1:80 or higher were considered as positive reactions.

Table 2. Reciprocal Titres of Antibodies to B. Burgdorferi S.L. in Wild Boars Determined by Indirect Haemagglutination Assay (IHA) Titre Number of examined

Total positive

180

1160

1320

1640

642

82

61

11

9

1

Total

The highest seropositivity rate was observed in the administrative regions MS (25.0%), PB (25.0%) and KH (24.1%), i.e., in northern Moravia, Silesia and eastern Bohemia, while a lower seroprevalence was recorded in the regions PZ (16.7%), OC (13.3%), JM (12.8%), VY (11.1%), JC (11.1%), ZL (10.3%) and LB (8.9%) (Table 1, Fig. 1). When analyzed statistically, prevalence rate of Bb antibodies in wild boars shot in KH and PB regions was significantly higher than that from VY region (2 4.404; P 0.039). Similarly, seroprevalence of wild boar from KH and PB regions was significantly higher that in JC region (2 4.008; P 0.046). Differences of all other pair-wise comparisons were insignificant (Table 3). A significantly lower overall seroprevalence was recorded in winter (4.2% to 9.7%) and in spring (17.4% to 24.3%) than in summer (33.3%). A gradual increase of seropositive wild boars was noticed between March and May, from 17.4% to 46.7% (Table 4). Discussion

Statistical analysis Chi-square test was used to estimate the association between categorical data in 2x2 contingency tables (SOLO statistical program, BMDP Statistical Software, Los Angeles, California, USA). For data analysis, collection sites were clustered into administrative and physiogeographic regions. Results Serologic results confirmed wild boars’ exposure to Bb at widely separated sites. Antibodies to Bb were detected in all ten administrative regions. Overall, positive reactions were established in 82 (12.8%) of the 642 examined animals (Table 1). The antibody titres of seroreactive animals varied from 1:80 to 1:640 (Table 2).

Sensitivity and specificity of IHA was demonstrated to be comparable or superior to those of the indirect fluorescence assay (IFA) (Jur˘ icová et al. 2002). It was further found that IHA compared with the standard IFA is quicker, easier to perform, more economical (saving sera and antigen) as well as better readable. The IHA test used includes antigens of all three main human pathogenic genomic species occurring in Europe, viz Borrelia garinii, B. afzelii and B. burgdorferi s.s., but differentiation of antibodies against those particular species is impossible by this assay. The records of antibodies to Bb among wild boars correspond to a few previous reports in the Czech Republic. For instance, the antibodies were found in three out of five wild boars in South Moravia (Treml et al. 1994). During serolog-

Table 3. Statistical Comparison of the Differences in Antibody Prevalence to B. Burgdorferi S.L. in Wild Boars Between Individual Regions. Values of X2 3.841 Indicate Significant Differences (P 0.05), Printed in Bold Regions compared V1* vs. V2 V3 V4 V5 V6

2 0.796 0.169 0.142 3.678 0.168

Regions compared

2

V2 vs. V3 V4 V5 V6

1.270 1.160 0.605 1.022

Regions compared

2

V3 vs. V4 V5 V6 V4 vs. V5 V6 V5 vs. V6

0.000 4.404 0.015 4.008 0.014 3.185

Explanation: *V1 regions JM ZL; V2 regions MS OC; V3 region VY; V4 region JC; V5 regions KH PB; V6 region LB PZ

ANTIBODIES TO BORRELIA BURGDORFERI S.L. IN WILD BOARS

481

Table 4. Anti-Borrelia Antibodies in Wild Boars by Season (X2 40.118; D.F. 6; P 0.001) Month January February March April May June July Total

No. of examined 48 299 23 37 15 39 12 473.*

No. of positive

Seropositivity (%)

2 29 4 9 7 13 4 68

4.2 9.7 17.4 24.3 46.7 33.3 33.3 14.4

*Other animals of unrecorded date of sampling are not included.

ical examination in the same place, Jur˘ icová et al. (1996, 2000) detected positivity to Bb in 18.2% and 25.3% of wild boars. On the contrary, incidence of antibodies in the wild boar was higher (40%) in the central part of Bohemia (Zeman and Janus˘ka 1999). The results in other European countries vary. In France, Doby et al. (1991) and Perez-Eid (1995) detected antibodies in 19–20% of wild boars. A much higher seroprevalence to Bb (41.6%) was found in Bulgaria (Angelov et al. 1995), while sera of all 10 examined wild boars from Croatia were negative (Golubic et al. 1998). Our study shows that antibodies to Bb are common among wild boars collected in all examined regions of the Czech Republic. In total, 12.8% of examined animals (8.9–25.0% according to the region) showed Bb antibodies. No statistically significant difference was observed in seroprevalence rate recorded among different regions, except for VY and JC regions, where significantly lower antibody rates were found as compared with KH and PB regions. Geographical distribution of Bb in the Czech Republic correlates with the presence of the vector tick I. ricinus, which is prevalent in appropriate habitats of all territories of Czechland, with minor differences between individual localities (Cˇerny´ 1972). Previously, extensive studies were carried out and borreliae were detected in larval, nymphal, and adult

stages of I. ricinus ticks in many regions of the Czech Republic (Hubálek et al. 1971; Halouzka et al. 1995; Hubálek and Halouzka 1998; Zeman 1998; Zeman and Daniel 1999; Danielová et al. 2004). Seasonal differences in the seroprevalence of wild boars were observed in the study. The increasing trend of prevalence was observed from March to July, with the peak recorded in May (46.7%). This trend correlates with increasing numbers and activity of I. ricinus tick between March and May (Hubálek et al. 1994). Evidence of IgG antibodies in wild boar in the Czech Rebublic can only serve as an indicator of mere contact with the agent of LB, but it will not determine whether a titre represented current or past infection. It is known that the antibody response to Bb in experimentally infected deer takes three weeks to develop and appears to be decreasing by 10 weeks (Lane et al. 1994; Luttrell et al. 1994). Tuomi et al. (1998) found that humoral immune responses to Bb in experimentally infected cattle appeared 14 to 51 days after inoculation. Titres of Borrelia antibodies in cattle in endemic areas persist as a result of frequent reinfections (Parker and White 1992). No similar experiments were performed on the wild boar. Seropositivity does not mean that wild boars are spirochetemic and infectious. Generally, the wild boar has some importance in the ecology and dynamics of the circu-

FIG. 1. Geographic distribution of regions in the Czech Republic from which the wild boars were sampled, and seroprevalence rate (%) of wild boars to Borrelia burgdorferi s.l. For the abbreviations of regions, see Materials and Methods.

482 lation of Bb spirochetes in Europe, especially as a host of vector ticks, or as a potential host of ticks that could infect themselves by co-feeding (Gern and Rais 1996; Ogden et al. 1997). In conclusion, serologic testing of wild boar sera could be one of the surveillance methods for determining the presence or absence of the LB agent in different areas. Knowledge of Bb circulation in animal populations helps to detect new foci of this disease, thus contributing to risk assessment of Lyme borreliosis. Acknowledgments Sera of wild boars were kindly supplied by the State Veterinary Institute in Jihlava (the former director MVDr. J. Holejsˇovsky´, MVDr. P. Barták and MVDr. I. Kucharovic˘ová) and Veterinary and Pharmaceutical University, Faculty of Veterinary Medicine in Brno (Professor MVDr. F. Treml). We wish to thank Mr. J. Pes˘ko for his technical assistance. Supported by the Grant Agency of the Czech Republic (Grant No. 524/96/1059) and EU grant GOCE-2003-010284 EDEN; it is catalogued by the EDEN Steering Committee as EDEN0108 (www.eden-fp6project.net/). Disclosure Statement No competing interests exist. References Angelov, L, Arnaudov, D, Rakadjieva, T T, Lichev, D, Kostova, E. Lyme borreliosis in Bulgaria (epidemiologic and epizootologic review). In: Report of WHO Workshop on Lyme Borreliosis Diagnosis and Surveillance. Warsaw, Poland; 1995: 41–52. Cˇerny´, V, The tick fauna of Czechoslovakia. Folia Parasitol 1972; 19:87–92. Danielová, V, Daniel, M, Rudenko, N, Golovchenko, M. Prevalence of Borrelia burgdorferi sensu lato genospecies in hostseeking Ixodes ricinus ticks in selected South Bohemian locations (Czech Republic). Centr Eur J Public Hlth 2004; 12:151–156. Doby, JM, Betremieux, C, Rolland, C, Barrat, J. The large forest mammals reservoir for Borrelia burgdorferi, agent of the Lyme disease? Serological examination of 543 deers and wild boars. Rec Méd Vét 1991; 167:55–61. Gern, L, Rais, O. Efficient transmission of Borrelia burgdorferi between cofeeding Ixodes ricinus ticks (Acari: Ixodidae). J Med Entomol 1996; 33:189–192. Gray, JS, Kahl, O, Janetzki, C, Stein J. Studies on the ecology of Lyme disease ina deer forest in County Galway, Ireland. J Med Entomol 1992; 29:915–920. Gray, JS, Kahl, O, Janetzki, C, Stein, J, Guy, E. The spatial distribution of Borrelia burgdorferi-infected Ixodes ricinus in the Connemara region of County Galway, Ireland Exp Appl Acarol 1995; 19:163–172. Halouzka, J, Jur˘icová, Z, Mátlová, L, Hubálek, Z. Borreliae in larval Ixodes ricinus ticks. Med Vet Entomol 1995; 9:205–206. Holubic, D, Rijpkema, S, Tkadlec-Makovec, N, Ruzic, E. Epidemiologic, ecologic and clinical characteristics of Lyme borreliosis in northwest Croatia. Acta Med Croatica 1998; 52: 7–13.

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