European Journal of Epidemiology 16: 869±873, 2000. Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Detection of spirochaetes of Borrelia burgdorferi complexe in the skin of cervids by PCR and culture Pichon Bruno1, Gilot Bruno2 & PeÂrez-Eid Claudine1
UniteÂ d'EÂcologie des SysteÁmes Vectoriels ± Institut Pasteur, 25 rue du Dr Roux, Paris and 223 AlleÂe des AubeÂpines, Villeneuve les Avignon, France
Accepted in revised form 11 December 2000
Abstract. To determine whether deer may play a role in the cycle of the Lyme disease spirochete Borrelia burgdorferi, we sought evidence for the presence of the pathogen in skin of deer and roe deer. Biopsies of 2 mm3 were taken at four dierent levels from nail to tarsus. A total of 50 animals (200 biopsies) were shot in the Lyme disease foci of Rambouillet during the hunting season 1995±1996 and 1996±1997, from the begining of November to the end of February. Borrelia burgdorferi s.l. DNA was detected by PCR in 18 biopsies from 14 animals (28%). Borrelia burgdorferi s.str. was predominant (50%), followed by B. garinii (30%) and B. afzelii (10%). Multiple
infections were detected in four animals: same species at dierent levels or two dierent species from the same biopsy or from dierent biopsies from the same foot. A total of 125 biopsies were cultivated on BSKH medium. Cultures at 160 days revealed immobile spiralled forms in 10 cultures. One, from a deer killed at the end of December, was con®rmed by PCR as B. burgdorferi s.str. These results, frequency of detection of spirochetes by PCR in the skin, multiple infections and alive spirochetes in biopsies taken out side the season of activity of ticks strongly suggest an anity of Lyme disease spirochetes for skin of cervids.
Key words: Bacteriology, Epidemiology, Lyme disease, Public health, Reservoir, Ticks
Introduction The Lyme disease spirochete Borrelia burgdorferi sensu lato (s.l.) is maintained in wild zoonotic cycles involving dierent vertebrates and is transmitted obligatorily by ticks of the genus Ixodes. These wild vertebrates constitute the vertebrate reservoir of the bacterium. A vertebrate acting as a reservoir is a susceptible vertebrate, able to amplify the pathogen more or less. It acts as ampli®er when it increases the level of infection in the vector population, and as conservator when it maintains the pathogen during conditions unfavorable to transmission. The reservoir role of micromammals is accepted by both European [1, 2] and North-American authors [3, 4]. By contrast the role of large mammals, particularly cervids, remains controversial. The frequency of antibodies in their sera indicates that deer are frequently exposed to infected ticks. The prevalence of seropositive deer ranges from 4 to 45% in Europe (in France: [5±7]), from 2 to 61%  in United States and in Japan the average seroprevalence is 25%, but it can reach 75% in summer . According to a majority of investigators, cervids do not act as an eective reservoir [10±13]. According to a minority they could possibly have a role [14±16]. In previous studies we observed a similar prevalence of infested Ixodes ricinus nymphs in areas with
high density of deer and roe deer (100 animal/ha) and low density of small mammals and in areas with low density of red deer and roe deer (20 animals/ha) , which led us to search for additional reservoirs to small mammals. In parallel, recent studies concluded that roe deer were heavily parasitized by larvae and nymphs of I. ricinus in France . Similar observations were made on deer Cervus elaphus , and on roe-deer Capreolus capreolus . Materials and methods The Rambouillet Forest is located about 60 km south-west of Paris. Its total surface area is about 22,000 ha. It is an acidophilous oak wood typical of the Paris region with two main types of vegetal landscapes: the ®rst one is characterised by a forest cover, the second one by heath with sparse patches of wood and moor. The study was conducted in the forested landscape, which is highly favorable to I. ricinus. It includes several plant communities including pedunculate oak and hornbeam forest, pedunculate oak forest and Molinia, oligotrophic sessile ¯ower oak forest, high reed beds, and acidophilous willow forest. Biopsies were made on cervids killed by hunters, members of FeÂdeÂration de Chasse de la ForeÃt de
870 Rambouillet (Hunting Federation of the Rambouillet Forest), during the 1995±1996 and 1996±1997 hunting seasons, from the begining of November to the end of January. All biopsies were taken from the posterior foot, from the hoof to the tarsus. This location was preferred to the head because data have been published on the presence of I. ricinus larvae on the feet of roe-deer  and no data have been published on the presence on the head, in contrast to what is known for the adult and nymphal stages. Moreover feet are easier to obtain from hunters than other parts of the animal. That is important for current epidemiologic studies. The biopsies were 2 mm3 in volume. They were sampled at four levels along the foot, A±D, from the hoof to the tarsus. Sample A was taken in the immediate vicinity of the hoof, the other samples were taken at 5-cm intervals.
CGGTTCTGGAAC-3¢; 5¢-GTAACTTTCAATGTTGTTTTGCCG-3¢), and B. afzelii (primers GIII: 5¢TAAAGACAAAACATCAACAGATGAAATG-3¢; 5¢-TTCCAATGTTACTTTATCATTAGCTACTT3¢). PCR reactions were made in a 50 ml volume with 5 ml sample DNA solution and 45 ml reactive medium: 1.5 units of Taq polymerase (CETUS±La Roche), PCR buer 1X, 2.5 mM MgCl2, dNTP 200 mM of each base, 0.4 mM of each primer. Thermal cycles were made in a programmable thermocycler Perkin-Elmer 9600. The ampli®ed products were analysed by gel electrophoresis on 1.6% agarose stained with 0.1 mg ethidium bromide per ml of gel. Speci®city of the ampli®cation was con®rmed by DNA/DNA hybridisation using a previously described technique .
Research of B. burgdorferi group
(A) Isolation by culture The biopsies were put in culture no more than 7± 8 hours after the death of the animal. Each biopsy was shredded with a single-use scalpel on a sterile glass slide, then seeded into a Falcon tube with 4 ml of BSK-H (Sigma) medium, complemented with 8% rabbit serum and antibiotics (Rifampicin 50 mg/ml, Phosphomycin 20 mg/ml and Amphotericin B 2.5 mg/ml). The cultures were incubated at 34 °C. They were observed against a black background with a microscope at 400´ magni®cation after 8, 20, 40, and 160 days.
Fifty cervid feet were collected and analysed during the present study (Table 1) giving a total of 200 biopsies. Borrelia burgdorferi s.l. DNA was detected in 18 biopsies from 14 animals, which corresponds to a 28% infection prevalence. The infection prevalence in the cervids killed was 37.5 and 23.5% during the 1995±1996 and 1996±1997 hunting seasons, respectively, which is not statistically dierent (p 0:3295) (Fisher test). All three cervids species, C. capreolus, C. elaphus, and C. sika, were infested with Borrelia, with infection percentages varying from 14 to 40%. Three of the eighteen biopsies were located within 5 cm of the hoof (A or B biopsies), while the other 15 were located at more than 10 cm from the hoof (C or D biopsies). Computation of reduced deviation for paired series indicates that this localisation of positive biopsies is signi®cant (e 2.6680, p < 0:001) (comparison by sign test).
(B) Gene ampli®cation The cultures were centrifuged twice, each time for 20 min at 15,000 rpm, then washed with PBS 1X. The sediment was resuspended in PBS 1X and heated for 10 min at 100 °C. The biopsies were very ®nely shredded with a single-use scalpel and put into a Eppendorf tube with 300 ml lysis buer [Tris±HCl (10 mM); EDTA (0.5 mM); Tween 20 (0.5%); Nonidet P40 (0.5%); Proteinase K (0.3 mg/ml)] and incubated for 4 hours at 56 °C. During lysis, the tubes were re-homogenised at 30 min interval. After lysis, they were heated for 15 min at 100 °C, then centrifuged for 10 min at 10,000g. The supernatant was puri®ed using the Genclean II (Bio 101) kit. (C) Ampli®cation of the OspA gene The ampli®cation of the DNA of B. burgdorferi group was carried out according to the technique previously described . This technique is used to identify the three species currently known to be pathogenic for man, B. burgdorferi s. str. (primers GI: 5¢-AACAAAGACGGCAAGTACGATCTAATT-3¢; 5¢-TTACAGTAATTGTTAAAGTTGAAGTGCC3¢), B. garinii (primers GII: 5¢-TGATAAAAACAA-
Table 1. Frequency of infection with Borrelia burgdorferi s.l. in cervids shot during the hunting season 1995±1996 and 1996±1997 in Rambouillet forest Species
No. of animals
C. capreolus C. elaphus C. sika
Nov±Dec 1995 Nov±Dec 1995 Nov±Dec 1995
1 10 5
0 4 (40.0%) 2 (40.0%)
6 5 3 5 11 4
0 0 2 1 2 3
Total C. capreolus C. elaphus
Nov 1996 Dec 1996 Jan 1997 Nov 1996 Dec 1996 Jan 1997
(66.7%) (20.0%) (18.2%) (75.0%)
871 Table 2. Genospeci®que identi®cation of Borrelia burgdorferi s.l. in cervids collected during the hunting season 1995 Designation Species CEF1 CEF3 CEF5 CEF8 CS13 CS16
C. C. C. C. C. C.
elaphus elaphus elaphus elaphus sika sika
Biopsy Biopsy A C ± ± ± ± ± ±
± B. b NI B. g B. b + B. g. B. b B. b B. b B. a ± B. g ±
B. a: B. afzelli; B. b: B. burgdorferi sensu stricto; B. g: B. garinii; NI; strain unidenti®ed.
Genospeci®c identi®cation of the Borrelia species found in the tested samples (Table 2) revealed that the three pathogenic species for man were present. B. burgdorferi s.str. was predominant (50%), followed by B. garinii (30%) and B. afzelii (10%). One Borrelia sample could not be identi®ed. Genospeci®c identi®cation also showed multiple infections in four animals (CEF3, CEF5, CEF8, and CEF30). In some cases, the same species was observed at two dierent levels of the foot (CEF5, CEF8, levels C and D), in other cases two dierent species were identi®ed from the same foot (CEF3 and CEF5) or even from the same biopsy (CEF5, biopsy C). A total of 125 biopsies were cultivated on BSK-H medium. Observations made at 8, 20, and 40 days were all negative. Observations at 160 days revealed immobile spiralled forms in 10 cultures (8%). The infection prevalence per biopsy was therefore the same whether detected by culture (8%) or by PCR (9%). In one of these cultures (CEM48C), from a deer killed on 30 December, a Borrelia species of the burgdorferi group was con®rmed by PCR; it was identi®ed as B. burgdorferi s. str. Discussion The present results concern the actual presence of the B. burgdorferi s.l. in deer and not just the presence of antibodies related to its infection as carried out in many studies. These Borrelia were taken from parts of the body known to be feeding sites for the larvae and nymphs of I. ricinus. Borrelia DNA was found in 28% of specimens of the three species of cervids, Cervus elaphus, Cervus sika and Capreolus capreolus. All three pathogen genospecies were present with B. burgdorferi s. str the most frequent: 50% B. burgdorferi s. str., 30% B. garinii, 10% B. afzelii. Survival of spirochetes was also observed by isolation in BSK medium. Biopsies were made from four dierent foot levels. Borrelia DNA is statistically more often detected in the part far of the hoof. This level is possibly the preferred zone of ®xation of larvae and nymphs, although this cannot be con®rmed. (It could not be
proved either in a previous study demonstrating a heavy infestation of roe deer by larvae and nymphs of I. ricinus because of inadequacy of the sampling method used during that study .) Three types of infection were observed: two with two dierent species, either in the same biopsy or in two dierent biopsies from the same animal. The third type of infection was with the same species in dierent parts of the foot. The presence of two genospecies of bacterium in one biopsy may explain the poly-infection of some ticks [17, 18, 20, 21]. The origin of infection with the same genospecies in two dierent levels of the same foot may be due to two dierent ticks or to a single one with spirochete diffusion through the skin. Isolation of Borrelia, outside the season of activity of ticks (March±October), suggests that spirochetes survive for several weeks as a persistent and localised infection in the skin of cervids (close examination of the biopsy sites failed to show any trace of recent tick bites). The genospecies identi®ed was B. burgdorferi s. str. It is dicult to determine whether or not this re¯ects a predominant presence of this species in cervids. It is well known that B. burgdorferi seems to grow better than the other species on commercial BSK II medium, but B. burgdorferi was the species identi®ed by culture and the one most often identi®ed by PCR. Cervids have been considered as reservoirs of B. burgdorferi because spirochetes have been isolated from their blood in Europe as in United States [22±24] and their sera react with antigens of these spirochetes. However to function as a competent reservoir a vertebrate must be capable of infecting feeding ticks with spirochetes. Studies on the infectivity of cervids for ticks remain controversial. Some observations suggest that deer  and roe deer  do not infect ticks or that deer sera have an indiscriminatory borreliacidal activity against all the genospecies tested . Some others observations suggest the contrary: experimentally inoculated deer can serve as a source of at least two geographic strains of B. burgdorferi to I. scapularis larvae and nymphs for at least several weeks , higher infection prevalence in ticks taken from deer ears than in questing population are reported  and infection rate of I. ricinus nymphs are not signi®cantly dierent between areas with high density of deer (>100/ha) and areas with low density (<20/ha) . In this context, the results presented here do not settle the controversy. However our study shows that cervids can harbour spirochetes in their skin, as is known for humans. Thus, this particular localization is not only consistent with the presence of spirochete on ticks feeding sites but also with the presence of alive spirochetes in a biopsy taken at the end of December, a period of no tick activity, and with multiple infections with two genospecies. The presence of alive spirochetes in deer skin could facilitate
872 the transmission of Borrelia to ticks during their blood meal. The role of cervids in supporting tick population has been demonstrated in many studies [25, 26]. The present work points out the occurrence of live spirochaetes in the skin of deer feet some time after the end of the annual tick feeding season. The competence of deer as transmission hosts is not yet proven but these data will be useful to keep the debate alive and direct someone to do the de®nitive experiment, as xenodiagnostic, to test for transmission from infected ticks to uninfected ticks via naturally infected deer.
Acknowledgements We acknowledge Mr. Wildmer of the Division of Rambouillet Forest of The Oce National des ForeÃts for providing study sites and the hunters of The FeÂdeÂration de la Chasse and of The Oce National des ForeÃts for saving deer feet up. We are indebted to Edmond Godfroid (GeÂneÂtique AppliqueÂe ± UniversiteÂ Libre de Bruxelles) for providing B. burgdorferi group gene-speci®c primers. This investigation received ®nancial support in part by the Gould-Mathers Foundation (USA) and by grants from the French Ministry of Education and Research (MinisteÁre de l'Enseignement SupeÂrieur et de la Recherche) and the Recherche & Partage Association (Caisse d'EÂpargne).
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Address for correspondence: PeÂrez-Eid Claudine, UniteÂ d'EÂcologie des SysteÁmes Vectoriels ± Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France Fax: +33-140-613-089 E-mail: [email protected]