Apodemus species mice are reservoir hosts of Borrelia garinii OspA serotype 4 in Switzerland.

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.

Mitochondrial gene content, arrangement and composition compared in African and Asian schistosomes.

Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14 415 bp), S. japonicum (Anhui strain, China; 14 085 bp) and S. mekongi (Khong Island, Laos; 14 072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 aa), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79% for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species.

Transmission of Borrelia afzelii from Apodemus mice and Clethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and overwintering maintenance.

This study deals with the ecology of Lyme borreliosis in Europe. The relationships between Borrelia burgdorferi sensu lato spirochetes, Clethrionomys and Apodemus rodent reservoirs and the Ixodes ricinus tick vector were investigated during 16 consecutive months in an enzootic area in Switzerland. Cultivation of ear skin biopsies was used to isolate spirochetes from C. glareolus, A. sylvaticus, A. flavicollis and Glis glis. Borrelia infection was more frequently observed in Clethrionomys than in Apodemus. Tick xenodiagnosis was used to determine the infectivity of rodents. The infection rate in ticks fed on Clethrionomys was higher than that in ticks fed on Apodemus, but Apodemus yielded more infected ticks than Clethrionomys because of a better tick moulting success. Xenodiagnostic ticks were placed into BSK medium to obtain isolates. Isolates from rodents and rodent-feeding ticks were all identified as B. afzelii. The follow-up of the infectivity status of repeatedly recaptured rodents clearly demonstrated that these hosts remained infective for ticks during winter till the following spring. Comparing C. glareolus and A. sylvaticus, each rodent species showed different host infection, different host infectivity and contributed differently to the moulting success of feeding ticks. These factors influence differentially the pattern of transmission of B. afzelii from Clethrionomys voles and Apodemus mice to I. ricinus ticks.

Relationship between Borrelia burgdorferi sensu lato species, red squirrels (Sciurus vulgaris) and Ixodes ricinus in enzootic areas in Switzerland.

The infection and reservoir status of red squirrels (Sciurus vulgaris) for Borrelia burgdorferi sensu lato were studied in Switzerland. B. burgdorferi sensu lato was isolated from 15 skin samples from 4/6 dead red squirrels, victims of road traffic. Isolates were identified using restriction fragment length polymorphism (RFLP): B. burgdorferi sensu stricto was present in 14 culture tubes containing skin samples and B. afzelii in two other tubes. A mixed infection was revealed in one case. A total of 227 ticks attached to squirrels were cultivated in BSKII medium and 90 isolates were obtained. Genotypic identification by RFLP showed that B. afzelii (59%) and B. burgdorferi sensu stricto (46%) dominated in ticks feeding on red squirrels. Data collected from one particular animal, highly infested with Ixodes ricinus and harbouring numerous Borrelia-infected Ixodes ricinus ticks, showed that transmission of B. burgdorferi sensu lato occurred from S. vulgaris to feeding ticks. More precisely, B. burgdorferi sensu stricto and B. afzelii were mainly transmitted from S. vulgaris to ticks. The present data emphasized the results obtained previously from small rodents and birds in Japan and in Switzerland, showing the occurrence of specific associations between host species and Borrelia genospecies.

An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes.

The reservoir competence of passerine birds for the Lyme borreliosis spirochetes was studied in an enzootic focus in Switzerland. Skin aspirates and skin biopsies were used to isolate Borrelia spirochetes from Turdus species. B. burgdorferi sensu lato was isolated and/or PCR-detected in BSK medium containing skin biopsy or skin aspirate from 5 blackbirds (T. merula) and one song thrush (T. philomelos). Seven isolates were obtained from 3 different blackbirds. Either B. garinii or Borrelia from the genomic group VS116 was found in bird skin samples. Mixed infection occurred in 2 cases. Tick xenodiagnosis was used to determine whether blackbirds transmitted Borrelia to ticks. Five xenodiagnoses were performed on 3 different blackbirds. Borrelia DNA was detected in BSK medium inoculated with xenodiagnostic ticks from all the passerines tested. Isolates cultured from xenodiagnostic ticks were obtained from 2 blackbirds. Isolates belonged to group VS116 (n = 10) and to B. garinii (n = 1). Our study has shown that Turdus sp. are infected by B. garinii and by Borrelia from group VS116 and that blackbirds are implicated as reservoirs for these 2 genomic groups of Borrelia, as they transmit living borreliae to ticks. An association seems to exist between birds and Borrelia VS116, and to a lesser extent, B. garinii, similar to the association existing between small rodents and B. afzelii. Our observations emphasize the fact that different enzootic cycles maintain Lyme borreliosis spirochetes in nature.

Natural history of Borrelia burgdorferi sensu lato.

Lyme borreliosis is a zoonosis: its causative agent, B: burgdorferi, circulates between ticks and a large range of vertebrates. Identification of the hosts which are responsible for the infection of the vectors is extremely important to determine the potential risk of infection in an habitat. Various small mammals and bird species are considered reservoirs for the Lyme disease spirochetes. Grey and red squirrels, hedgehogs as well as hares and rabbits can develop an infection and transmit B. burgdorferi sensu lato to feeding ticks. In Eurasian endemic areas, many different Borrelia species circulate between ticks and vertebrate hosts. Studies have shown that European and Asian genospecies are associated with specific groups of vertebrate hosts, such as B. valaisiana and B. garinii with birds, B. afzelii with small mammals and B. burgdorferi ss and B. afzelii with red squirrels. However, such associations are not always observed as in Japan where B. garinii, B. afzelii and unidentified Borrelia species are found in small mammals. Some enzootic cycles involving tick species which do not feed at all on humans or which rarely feed on humans have been described in Europe and USA. It is likely that many existing enzootic foci have yet to be discovered. The circulation of B. burgdorferi in silent foci does not have important implications for human health, but it demonstrates the complexity of the ecology of this microorganism and the variety of ecological niches this spirochete can occupy.

Apodemus sp. rodents, reservoir hosts for Borrelia afzelii in an endemic area in Switzerland.

Borrelia burgdorferi is maintained in nature in transmission cycles alternatively involving ticks and reservoir hosts. Small rodents like Apodemus mice and Clethrionomys voles are the primary reservoir of Lyme disease in Europe. In this study, we analyzed by SDS-PAGE and Western blot 20 borrelial isolates from xenodiagnostic ticks fed on four Apodemus sp. mice captured in the Staatswald forest (Switzerland). All isolates but one showed a homogeneous protein pattern expressing an outer surface protein, (Osp) A of 32 kDa and an OspB of 35 kDa and reacted with monoclonal antibody (mAb) I 17.3 specific for B. afzelii. One isolate expressed an OspA of 32.5 kDa and an OspB of 35 kDa and did not react with species-specific mAbs I 17.3, D6 and H3TS, but was shown to belong to B. afzelii by Southern blot analysis. The possibility exists that non-cultivatable borreliae are present in xenodiagnostic ticks. However, our results clearly show that Apodemus sp. are reservoir hosts for B. afzelii, since this genospecies is transmitted from Apodemus sp. to feeding larval ticks.

Strain variation of Lyme disease spirochetes isolated from Ixodes ricinus ticks and rodents collected in two endemic areas in Switzerland.

The relationship among Lyme borreliosis, Borrelia genospecies, rodent reservoirs, and Ixodes ricinus L. ticks was studied in two endemic areas in Switzerland. Ear punch biopsies and sampling of internal organs were used to isolate Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt and Brenner) from small mammals, Apodemus sylvaticus L., A. flavicollis Melchoir, Clethrionomys glareolus Schreber. Spirochetes were isolated from ear tissue and spleen of the rodents. Isolates were homogeneous and belonged to typing group II identified as B. afzelii (Canica, Nato, du Merle, Mazie, Baranton and Postic). Our data show that a specific association exists between B. afzelii and rodent reservoirs in European foci. Borreliae were also isolated from field-collected I. ricinus ticks from the same study areas. Proteinic and antigenic analysis indicated that more than one genospecies were present in the tick population. This suggests that other vertebrate hosts may serve as reservoirs of other Borrelia genospecies implicated with Lyme disease.

Identification of spotted fever group rickettsiae isolated from Dermacentor marginatus and Ixodes ricinus ticks collected in Switzerland.

When 155 ticks collected in different regions of Switzerland were tested by the hemolymph test, 10.3% were found to contain spotted fever group rickettsiae. Six rickettsial isolates were made from Dermacentor marginatus ticks and three were made from Ixodes ricinus ticks. The polymerase chain reaction followed by restriction fragment length polymorphism analysis showed that the Dermacentor ticks were infected with Rickettsia length polymorphism analysis showed that the Dermacentor ticks were infected with Rickettsia slovaca and the Ixodes ticks were infected with a spotted fever group rickettsia. Microimmunofluorescence serologic type, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins, and Western blot assay with polyclonal mouse antisera confirmed the results and determined that the Ixodes were infected with R. helvetica, the only previously described Swiss rickettsia. However, an additional new strain that could not be isolated was detected in one I. ricinus by hemolymph test and provisionally characterized by enzymatic restriction of its amplified DNA.

Borrelia burgdorferi in rodents (Apodemus flavicollis and A. sylvaticus): duration and enhancement of infectivity for Ixodes ricinus ticks.

Ixodes ricinus is an important vector of Borrelia burgdorferi in Europe, and small rodents (Apodemus flavicollis, A. sylvaticus and Clethrionomys glareolus) are important sources for infecting ticks. In this study, we examined their reservoir role by studying the duration of their infectivity for ticks. A. flavicollis and A. sylvaticus mice captured in nature were exposed to uninfected I. ricinus larvae at different times after their capture: 10 days, and 2, 7, 11, 14 and 40 months. Ticks were examined for spirochaetes after moulting using direct immunofluorescence. All animals remained infective for ticks their life long but the efficiency of transmission from hosts to ticks varied from one individual to the other, presenting a three-fold variation (26.5% to 81.4%). Rodents continously exposed to successive infestations by larval I. ricinus ticks over a period of one month showed an enhancement of infectivity for larval ticks during this period.

Borrelia burgdorferi in a focus of Lyme borreliosis: epizootiologic contribution of small mammals.

The contribution of woodmice (Apodemus sylvaticus), yellow-necked mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus) was compared in a focus of Lyme borreliosis in Switzerland during a 7 months' study. All three species of mice and one species of shrews (Sorex araneus) were shown parasitized by infected Ixodes ricinus immatures. About 14% of larvae and 50% of nymphs collected on small mammals were infected with B. burgdorferi. Spirochetes were isolated from blood of 3 woodmice and one yellow-necked mouse. The infectious status of rodents was estimated by tick xenodiagnosis. Prevalence of infected rodents ranged from 20% to 44%. Mice presented a higher potential infectivity than voles. The prevalence of infected rodents showed a seasonal variation.

Ixodes ricinus immatures on birds in a focus of Lyme borreliosis.

The infestation of birds by immature Ixodes ricinus was studied during 6 months in a Swiss woodland, where Lyme borreliosis is endemic. Thirteen passerine species were found to be parasitized by I. ricinus subadults and specially Turdus merula, T. philomelos and Erithacus rubecula. Overall, 300 larvae and 162 nymphs were collected on 95 avian hosts. Prevalence of infestation of nymphs on birds was higher in spring; larvae peaked in summer. The infection of birds by Borrelia burgdorferi was also studied using blood cultivation and examination of ticks. Motionless spirochetes were isolated from two E. rubecula. Infected ticks were removed from five species of passerines, and mainly three species of Turdidae (T. merula, T. philomelos and E. rubecula). Infection rate of larvae and nymphs by spirochetes averaged 16.3% and 21.7%, respectively. These percentages, compared to the infection rate of questing ticks collected through dragging, suggest that some Turdidae may play a role as amplifying hosts for spirochetes in the focus.