Free-living adult Ixodes ricinus L. were collected in Amdoun, situated in the Kroumiry mountains in northwestern Tunisia (North Africa). Using direct fluorescence antibody assay, the infection rate of field-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n = 72). No difference in infection rate was observed between male and female ticks. Spirochetes that had been isolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identified as Borrelia lusitaniae (formerly genospecies PotiB2). This is the first identification of a genospecies of Borrelia burgdorferi sensu lato from the continent of Afric

Aeschliman, Andre

Bouattour, Ali

Gern, Lise

Gharbi, Mohamed

Ginsberg, Howard S.

Hu, Chang Min

Zhioua, Elyes

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SHORT COMMUNICATIONInfection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferisensu lato in North AfricaELYES ZHIOUA,1 ALI BOUATTOUR,2 CHANG MIN HU,3 MOHAMED GHARBI,2ANDRE AESCHLIMAN,3 HOWARD S. GINSBERG,4 AND LISE GERN3J. Med. Entomol. 36(2): 216—218 (1999)ABSTRACT Free-living adult Ixodes ricinusL.were collected inAmdoun, situated in theKroumirymountains in northwestern Tunisia (North Africa). Using direct ßuorescence antibody assay, theinfection rate of Þeld-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n 5 72). Nodifference in infection rate was observed between male and female ticks. Spirochetes that had beenisolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identiÞed as Borrelialusitaniae (formerly genospecies PotiB2). This is the Þrst identiÞcation of a genospecies of Borreliaburgdorferi sensu lato from the continent of Africa.KEY WORDS Ixodes ricinus, Borrelia burgdorferi, Borrelia lusitaniae, TunisiaIN EUROPE, Ixodes ricinus L. is the principal vector ofBorrelia burgdorferi, the etiologic agent of Lyme bor-reliosis (Aeschlimann et al. 1986). Borrelia burgdorferisensu lato is now known to include at least 5 geno-species associated with I. ricinus: Borrelia burgdorferisensu stricto, Borrelia garinii, Borrelia afzelii, Borreliavalaisiana (formerly genospeciesVS116), andBorrelialusitaniae(formerlygenospeciesPotiB2)(Barantonetal. 1992; Canica et al. 1993; Postic et al. 1994, 1997; LeFle`che et al. 1997;Wang et al. 1997). The geographicaldistribution of I. ricinus covers southern Scandinavia,the British Isles, Central Europe, France, Spain, Por-tugal, Italy, the Balkans, East Europe, northern Iran,and North Africa (Perez and Rodhain 1977, Stanek etal. 1988). Infection of I. ricinus by B. burgdorferi sensulato has been reported from many European coun-tries. In Africa, however, primarily serological andclinical results have been reported (Stanek et al. 1986,Ginsburg et al. 1991, Zahaf et al. 1994, Mhalu andMatre 1996), and fewentomological studies havebeenperformed (Zhioua et al. 1989). The objectives of thisstudy were to determine the infection rate of I. ricinusby B. burgdorferi sensu lato at a sample site in North-ern Africa (Tunisia), and to identify the genospeciesof Borrelia isolated from I. ricinus from Tunisia.Materials and MethodsStudy Site. In Tunisia, I. ricinus is localized in thenorthwest, mainly in the mountains of Kroumiry (At-las mountains), where it is abundant in oak forests(Zhioua et al. 1989, Bouattour and Darghouth 1996).These forests have rich undergrowth and harbor smallmammals, deer, and wild boar (Gharaibeh 1997). Theactivity peak of adult I. ricinus in this area is in Feb-ruary—March (Senevet and Rossi, 1924, Sergent andPoncet 1937, YousÞ-Monod and Aeschlimann 1986),so we sampled ticks in March 1996. Unfed adult tickswere collected byßagging or by direct collection fromthe grass in the region of Amdoun (Kroumiry).Spirochete Detection. Adults were surface-steril-ized and dissected to remove the gut. Midguts werechilled, placed on glass slides, and examined for thepresence of spirochetes using direct ßuorescent anti-body assay (DFA) (Zhioua et al. 1994). Antigensweredried for at least 2 h at 348C and subsequently Þxedwith acetone for 10 min. Preparations were staineddirectly with high-titered ßuorescein isothiocyanateconjugated anti-B. burgdorferi goat antibody (KPL,Gaithersburg, MD).In 1988, spirochetes were isolated from Þeld-col-lected I. ricinus from the region of Ain Drahim(Kroumiry, Tunisia) (Zhioua et al. 1989). Unfortu-nately, identiÞcation of these spirochetes was not pos-sible in 1988 because the cultures were contaminated(Zhioua et al. 1989), and the isolates were stored at2708C. Technical advances now allow characteriza-tion of these isolates. Borrelia isolates were identiÞedin 1996 by rrf (5S)-rrf (23S) spacer restriction frag-ment-length polymorphism (RFLP) analysis as de-scribed by Postic et al. (1994).Results and DiscussionIn total, 72 adult I. ricinus (41 females, 31 males)collected fromAmdoun in 1996were examined for thepresence of B. burgdorferi sensu lato by DFA. Theinfection rate was 30.5% (22/72). No signiÞcant dif-1 Center for Vector-Borne Disease, Woodward Hall, University ofRhode Island, Kingston, RI 02881.2 InstitutePasteur ofTunis,Department ofMedical Entomology, 13Place Pasteur, 1002 Tunis, Tunisia.3 Institute of Zoology, Emile Argand 11, University of Neuchaˆtel,Neuchaˆtel, Switzerland.4 USGS Patuxent Wildlife Research Center, Woodward Hall-PLS,University of Rhode Island, Kingston, RI 02881.ference was observed between the infection rates offemales (36.6%, n 5 41) and males (22.6%, n 5 31) (x25 1.63, df 5 1, P 5 0.2).IdentiÞcation of 2 strains of spirochetes isolatedfrom Tunisia in 1988 showed 1 genospecies, B. lus-itaniae (Fig. 1). Borrelia burgdorferi sensu stricto, B.garinii, B. afzelii, and B. valaisiana were not observed.Here,we report thepresenceofB.burgdorferi sensulato in I. ricinus on the African continent. On a sub-tropical island in the Atlantic, northwest of continen-tal Africa, the infection rate of I. ricinus by B. burg-dorferi was 1.3% (Matuschka et al. 1994). The 30.5%infection rate we observed in Tunisia is within therange reported from European countriesÑ13.8% inSpain (Mareuez and Constan 1990), 9.6% in Belgium(Martin et al. 1990), 5—50% in Switzerland (Aeschli-mann et al. 1986, Gern et al. 1992), 20% in the Neth-erlands (Rijpkema et al. 1994), 18% in Germany (Ma-tuschka et al. 1992), 13% in Slovenia (Strle et al. 1995),45% in Croatia (Rijpkema et al. 1996), and 12.4% inFrance (Zhioua et al. 1996). Additional studies areneeded to determine the infection rates of immaturesand to identify the reservoir hosts of B. burgdorferisensu lato in Tunisia.Borrelia burgdorferi sensu stricto is the only patho-genic genospecies currently known to be present inthe United States (Postic et al. 1994). In Eurasia, B.afzelii and B. garinii are widely distributed (Fukunagaand Hamase 1995, Postic et al. 1997). In Europe, 3genospecies associated with Lyme borreliosis havebeen recognized to dateÑB. burgdorferi sensu stricto,B. garinii, and B. afzelii. These genospecies have beenisolated from patients, rodents, birds, and I. ricinusfrom various areas in Europe (Humair et al. 1995,Olsen et al. 1995, Picken et al. 1996, Zhioua et al. 1996,Postic et al. 1997). Two additional genospecies havebeen observed in I. ricinus and identiÞed as B. val-aisiana and B. lusitaniae (formerly the genospeciesVS116 and PotiB2, respectively) (Postic et al. 1994,1997; Pe´ter et al. 1995; Rijpkema et al. 1995; Le Fle`cheet al. 1997; Wang et al. 1997). To date, only a fewisolates have been identiÞed as B. lusitaniae; 3 fromPortugal, 2 from Moldavia, 1 from Ukraine, 1 fromCzech Republic (Postic et al. 1994, 1997), and now 1from North Africa. The roles of B. lusitaniae and B.valaisiana in the etiology of Lyme borreliosis remainto be clariÞed.In western, central, and northern Europe, I. ricinusis commonly infected with various Borrelia genospe-cies, but rarely with B. lusitaniae, which appears to belimited to the southern part of the northern Hemi-sphere. Additional isolates from I. ricinus are neces-sary to describe the heterogeneity of spirochetes andthedistribution of the various genospecies in southernEurope and North Africa. In sub-Saharan Africa, theexistence of B. burgdorferi sensu lato remains unclear,but seems to be improbable because of the absence ofticks of the I. ricinus species complex (Mason et al.1994, Marjolet et al. 1995).AcknowledgmentsThe authors thank K. Dellagi (Director, Pasteur Instituteof Tunis) for his constant support, including logistics andlaboratory facilities. We also thank R. A. LeBrun, P. Logan,D. Dennis, and A. Spielman for constructive comments onearly drafts of the manuscript. Raquel Martinez and PaulJohnson provided technical assistance. This work beneÞtedfrom support by the Swiss National Science Foundation(Grant Nos. 3.975.87 and 32-29964.90), the U.S. NationalBiological Service, and the Department of Plant Sciences.This is Contribution No. 3446 of the College of ResourceDevelopment, University of Rhode Island.References CitedAeschlimann, A., E. Chamot, F. Gigon, J.-P. Jeanneret, D.Kesseler, and C. Walter. 1986. Borrelia burgdorferi inSwitzerland. Zentralbl. Bakteriol. Microbiol. Hyg. Ser. A263: 450—458.Baranton, G., D. Postic, I. Saint-Girons, P. Boerlin, J. C.Piffaretti, M. Assous, and P.A.D. Grimont. 1992. Delin-eation of Borrelia burgdorferi sensu stricto, Borrelia ga-rinii sp. nov., and Group VS461 associated with Lymeborreliosis. Int. J. Syst. Bacteriol. 42: 378—383.Bouattour, A., and M. A. Darghouth. 1996. First report ofBabesia divergens in Tunisia. Vet. Parasitol. 63: 161—165.Canica,M.M., F. Nato, L. duMerle, J. C.Mazie, G. Baranton,andD. 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Infection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferi sensu lato in North Africa

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