Apodemus Species Mice Are Reservoir Hosts of Borrelia garinii OspA Serotype 4 in Switzerland

Abstract

Among Borrelia burgdorferi sensu lato isolates, seven outer surface protein A (OspA) serotypes have been described: serotypes 1 and 2 correspond to B. burgdorferi sensu stricto and Borrelia afzelii, respectively, and serotypes 3 to 7 correspond to Borrelia garinii. In Europe, serotype 4 has never been isolated from Ixodes ricinus ticks until recently, although this serotype has been frequently isolated from cerebrospinal fluid from patients. In Europe, B. afzelii and B. burgdorferi sensu stricto were found associated with rodents and B. garinii was found associated with birds. In this study, the reservoir role of Apodemus mice for B. garinii OspA serotype 4 was demonstrated by xenodiagnosis. Apodemus mice are the first identified reservoir hosts for B. garinii OspA serotype 4.

Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, is a complex including at least 10 genospecies (1, 2, 4, 13, 14, 16, 17, 20, 23). Among them, three species are recognized as pathogenic for humans: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. In Europe, the tick Ixodes ricinus is the main vector of these pathogens.

Wilske et al. (24) defined seven outer surface protein A (OspA) serotypes among these three Borrelia species: serotype 1 corresponds to B. burgdorferi sensu stricto, serotype 2 corresponds to B. afzelii, and serotypes 3 to 7 correspond to B. garinii. Strikingly, B. garinii serotype 4 was never cultivated from I. ricinus until recently (10), although this serotype has frequently been cultivated from cerebrospinal fluid from patients from Germany, The Netherlands, Denmark, and Slovenia (18, 24, 25). In some areas of Europe where these species are indigenous, B. afzelii and B. burgdorferi sensu stricto are maintained by rodents and Borrelia valaisiana and B. garinii are maintained by birds (for a review, see reference 12). In the laboratory, it is difficult to obtain ticks infected by B. garinii from infected mice (3, 5). However, Hu et al. (10) obtained B. garinii serotype 4-infected ticks from laboratory mice. This finding, and the presence of B. garinii DNA in rodents and in xenodiagnostic ticks that fed on rodents in the eastern part of Europe (21; G. Khanakah et al., Abstr. VI Int. Conf. Lyme Borreliosis, abstr. PO77W, 1994), urged us to investigate whether rodents in Switzerland transmit serotype 4 to I. ricinus ticks.

From May to September 2000, small rodents were captured in three areas in Switzerland (Staatswald, Kernenried, and Langendorf) where Borrelia spp. are endemic. B. burgdorferi infection in rodents was monitored by tick xenodiagnosis. For xenodiagnosis, infection-free I. ricinus larvae from our laboratory colony (7) were placed on the head of each rodent, allowed to feed until repletion, and examined as nymphs for Borrelia spp. Each nymph was cleaned with 70% ethanol and then cut into two pieces. One half of the nymph was examined by immunofluorescence (IF) analysis using fluorescein isothiocyanate-conjugated polyclonal antibodies prepared from a pool of Lyme borreliosis patient sera and which detect all Borrelia species (6). The other half was used for Borrelia isolation as described by Gern et al. (6).

PCR and restriction fragment length polymorphism (RFLP) analysis of the rrf-rrl intergenic spacer were used for the identification of Borrelia species as described by Postic et al. (19). The pellet from 1 ml of spirochete growth culture (initial tube containing the tick) was washed, and DNA suspension was used as the template for amplification. The PCR products were analyzed by RFLP using MseI restriction endonuclease to identify the genospecies of B. burgdorferi sensu lato.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were performed as previously described (8), using monoclonal antibody (MAb) L32 1G3, which specifically reacts with the OspA of B. garinii serotype 4 (24).

The N-terminal end of the OspA shows a variable region of amino acids which allows the determination of serotypes (24). The sequences of ospA genes of various Borrelia serotypes (24, 26) were aligned, and the following primers in the 5′ end of the gene were designed: primer OspA-1F (5′-CAAAATGTTAGCAGCCTTG-3′) and primer OspA-3R (5′-GTGTATTCAAGTCTGGTTCC-3′). These primers allow the amplification of a DNA fragment of 389 bp, including the variable region described by Wilske et al. (24). Reaction volumes were 50 μl and contained 1.5 mM MgCl₂, 0.2 mM deoxynucleoside triphosphates, a 0.2 μM concentration of each primer, 1× Taq buffer, 1.25 U of Taq DNA polymerase (QIAGEN, Basel, Switzerland), and ultrafiltered H₂O. The PCR amplification was carried out for 35 cycles (denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for 1 min) with an initial denaturation step at 94°C for 2 min and a final extension step at 72°C for 10 min. The presence of PCR products was checked by using a 1% agarose gel and visualized by UV transillumination after ethidium bromide staining. PCR products were purified with a QIAquick PCR purification kit (QIAGEN). Cycle sequencing was performed by the dideoxy chain termination method using an ABI PRISM BigDye Terminator cycle sequencing kit (Applied Biosystems, Rotkreuz, Switzerland). Cycle sequencing parameters were as follows: denaturation at 96°C for 15 s, primer annealing at 53°C for 15 s, and extension at 60°C for 4 min. The cycle sequencing products were cleaned by ethanol precipitation and applied to an ABI PRISM 310 genetic analyzer (Applied Biosystems). DNA sequences were checked with Sequence Navigator software (Applied Biosystems). The identities of sequences were assigned by a search of the GenBank database.

A total of 29 rodents, captured in three areas where Borrelia is endemic and belonging to Apodemus sylvaticus (n = 24) and Apodemus flavicollis (n = 5), were subjected to xenodiagnosis. Seventeen mice (59%) transmitted spirochetes to xenodiagnostic ticks as determined by IF analysis and/or Borrelia isolation. Borrelia isolates (n = 27) obtained from xenodiagnostic ticks that fed on 10 individuals were characterized by PCR-RFLP analysis of the rrf(5S)-rrl(23S) intergenic spacer. B. afzelii was the dominant species among isolates from ticks that fed on mice (17 of 27, 63%), followed by B. garinii (10 of 27, 37%) (Table 1). Six out of 10 Apodemus mice transmitted only B. afzelii to xenodiagnostic ticks, whereas 3 individuals transmitted only B. garinii. One additional individual transmitted both species to xenodiagnostic ticks. The rates of B. garinii infection in xenodiagnostic ticks, determined by IF analysis, were as high as rates of B. afzelii infection (Table 1). Immunoblotting with MAb L32 1G3 revealed that all 10 B. garinii isolates belonged to OspA serotype 4. Sequencing of the amplified ospA gene confirmed the identification of species and serotypes obtained by PCR-RFLP and immunoblotting. ospA sequences of all B. garinii isolates matched the ospA sequence of serotype 4 type strain PBi. All mice which transmitted B. garinii serotype 4 to ticks came from one capture site only (Staatswald, Switzerland) (Table 1).

TABLE 1.

Borrelia infection rate of xenodiagnostic ticks that fed on Apodemus mice and characterization of Borrelia isolates from these ticks

Animal no.	Rodent species	Swiss capture site	Infection ratea	Characterization of isolates (n)b
1	A. sylvaticus	Staatswald	9/10	B. garinii, OspA type 4 (1)**
2	A. sylvaticus	Staatswald	9/10	B. garinii, OspA type 4 (6)
3	A. sylvaticus	Staatswald	6/10	B. garinii, OspA type 4 (2)
4	A. flavicollis	Staatswald	2/10	B. afzelii (1), B. garinii, OspA type 4 (1)
5	A. sylvaticus	Staatswald	9/10	B. afzelii (2)
6	A. flavicollis	Kernenried	9/10	B. afzelii (5)
7	A. sylvaticus	Kernenried	8/10	B. afzelii (2)
8	A. sylvaticus	Kernenried	6/10	B. afzelii (4)
9	A. sylvaticus	Langendorf	2/10	B. afzelii (2)
10	A. sylvaticus	Langendorf	8/10	B. afzelii (1)

^aNumber of ticks infected relative to total number of ticks examined, determined by IF analysis.

^bDetermined by PCR-RFLP, ospA sequencing, and Western blotting. n, number of isolates.

Serotyping of European isolates allowed us to distinguish seven OspA serotypes: serotypes 1 and 2 correspond to B. burgdorferi sensu stricto and B. afzelii, respectively, whereas serotypes 3 to 7 are included in B. garinii (24). Interestingly, the role of ticks as vectors of B. garinii serotype 4 has only recently been demonstrated (10). It was shown that I. ricinus nymphs collected in the field can transmit serotype 4 to laboratory mice and that these mice transmitted serotype 4 to xenodiagnostic ticks (10). This was surprising because B. garinii is usually associated with birds in nature (see reference 12) and because ticks infected by B. garinii are difficult to obtain from mice infected in the laboratory (3, 5). Since the association of B. garinii with rodents has been reported occasionally in Europe (21; Khanakah et al., Abstr. VI Int. Conf. Lyme Borreliosis, 1994.), we investigated the role of rodents as reservoirs for B. garinii serotype 4. B. afzelii was the Borrelia species most frequently transmitted from Apodemus mice to xenodiagnostic ticks. This confirms previous observations describing rodents as reservoir hosts for B. afzelii in Switzerland (9, 11). In the present study, rodents from one of the three studied areas transmitted B. garinii serotype 4 to xenodiagnostic ticks, whereas rodents from the two other areas transmitted only B. afzelii to ticks. This demonstrates that Apodemus mice can act as reservoirs for B. garinii serotype 4. Interestingly, the association of B. afzelii with B. garinii serotype 4 observed here in xenodiagnostic ticks corroborates previous observations (10). A mixture of B. garinii type 4 and B. afzelii was also isolated from the cerebrospinal fluid of a patient (24). This indicates that B. garinii serotype 4 may be present in mixed infections in rodents, humans, and ticks in nature, and that serotype 4 may be overgrown in culture by other types, possibly B. afzelii, which is more easily isolated from ticks (6). This could explain the previous difficulty in obtaining OspA serotype 4 isolates from ticks collected in the field (25). The fact that mice serve as a reservoir for B. garinii serotype 4 and not other B. garinii serotypes might be explained by serum resistance (10, 15). Interestingly, serotype 4 was resistant to human serum whereas the other B. garinii subtypes were sensitive (22).

In conclusion, the present study identified Apodemus mice as reservoirs of B. garinii serotype 4 in Switzerland.