Identification of a New Borrelia Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic
Horacio Gil1,2,†, Marta Barral1, Raquel Escudero2, Ana L. García-Pérez1 and Pedro Anda2,*
Journals. ASM.org
18 Okt. 2004
http://aem.asm.org/content/71/3/1336.fullABSTRACT
The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.
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However, new species of Borrelia transmitted by hard ticks (family Ixodidae) have been classified closer to the RF group: “Borrelia lonestari” (proposed name),
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This latter genospecies seems to have a larger distribution area, since related species have been found in Europe in I. ricinus (26, 67) and in America in Ixodes scapularis (74).
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In southern Spain, a new Borrelia species has been isolated from patients and soft ticks (3) in areas where RF is endemic (5, 14, 72). Moreover, in the north of Spain there are areas where LD is endemic that coincide with the distribution of I. ricinus (19). In these areas, several series of LD cases have been described (2, 33, 63),
and epidemiological studies of B. burgdorferi sensu lato in questing ticks (9), in ticks collected from animals (24), and in ticks collected from humans (25) have been performed. Since the first isolation of B. burgdorferi sensu lato in Spain (29), only a few isolates have been obtained (9, 62) and their characterization has shown a wide genospecies diversity and virulence in a mouse model (23). In the Basque country, our study region, cases of Lyme disease in humans have been reported; a serological survey showed 25% prevalence in outdoors workers, with antecedents compatible with LD in 15% of those who were seropositive (4). Moreover, our previous data confirmed the wide distribution of the vector I. ricinus and B. burgdorferi sensu lato in several areas of the Basque country (9).
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The position of R57 in both trees was identical, as an ancestor of both LD and RF Borrelia. Moreover, PCRs for 5S-23S rRNA, ospA, flaB, rpoB, and p66 yielded negative results, suggesting abundant sequence differences between this agent and other Borrelia species.
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The major reservoir hosts for B. burgdorferi sensu lato in the Basque country are still to be identified. The suitable candidates, with demonstrated roles as reservoirs elsewhere, are birds (39, 46, 68), dormice (56, 57), and squirrels (20, 38).
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Four different genospecies have been found infecting questing ticks, the most abundant being B. burgdorferi sensu stricto, followed by B. valaisiana and B. garinii. B. afzelii was infrequently founding infecting questing ticks. This is different from other European studies (37, 70
B. afzelii was infrequently founding infecting questing ticks. This is different from other European studies (37, 70), especially those from Central Europe, where B. afzelii is highly prevalent. B. afzelii seems to be maintained in nature mainly by a tick-small mammal cycle that does not include the involvement of birds (36, 40, 66). The fact that small mammals are not playing a relevant role as reservoir hosts for B. burgdorferi sensu lato in the study areas may explain the low prevalence of B. afzelii found, similar to that in Ireland (32), where B. valaisiana was highly prevalent and birds seemed to play a relevant role as reservoir hosts for this genospecies (39, 50). B. valaisiana was also prevalent in our region, suggesting that birds may also play a role in maintaining this genospecies in our area.
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In conclusion,
we have shown that although small mammals are not important reservoir hosts for B. burgdorferi sensu lato in areas of LD endemicity in the Basque country, they are maintaining a new spirochete species. This new species is widely distributed among small mammals within the study area; produces a disseminated infection that involves the skin, brain, and urinary bladder; and is thought to induce antibodies that cross-react with B. burgdorferi sensu lato. Signature nucleotides in the 16S rRNA sequence enabled us to design a diagnostic method that will be useful in determining the actual distribution of this agent in the area.
