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Infection and Immunity, September 2002, p. 4851-4858, Vol. 70, No. 9
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.9.4851-4858.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vitro Culture of Borrelia garinii Results in Loss of Flagella and Decreased Invasiveness

Ricela E. Sellek,1 Raquel Escudero,1 Horacio Gil,1 Isabel Rodríguez,1 Escolástica Chaparro,1 Esperanza Pérez-Pastrana,2 Amparo Vivo,2 and Pedro Anda1*

Servicio de Bacteriología, Centro Nacional de Microbiología,1 Servicio de Microscopía Electrónica, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220-Majadahonda, Madrid, Spain2

Received 28 December 2001/ Returned for modification 6 March 2002/ Accepted 6 June 2002

A virulent, low-passage culture of a tick-derived strain of Borrelia garinii was subjected to serial in vitro passages, from which inoculations were made into C3H/HeN mice. A full display of pathogenicity was observed through passage 4, as measured by cultures of ear punch biopsy samples and internal organs and determination of tibiotarsal joint swelling. Decreased dissemination through skin and infection of internal organs were observed beginning at passage 6. These losses correlated with both the selection of clones harboring 21% less flagella than the parent strain, as seen by electron microscopy, and loss of the motility of the higher passages, as demonstrated by a swarm assay. However, during the chronic phase (3 months after infection), spirochetes were cultured from the bladder and kidney of a mouse inoculated with passage 12. The kidney isolate had the same number of flagella and motility as the original low-passage isolate. Although we can't exclude the possibility that other subtle variations may be arising given the uncloned nature of the isolate, we have found a strong association between loss of flagella and decreased invasiveness. Arthritogenicity progressively decreased with passages, so that only 12.5% of chronically infected mice inoculated with passage 29 still presented with joint swelling, concurrent with a decrease in the staining intensity in a Southern blot with a vlsE-based probe. These results suggest a multifactorial model in which the number of flagella drives the invasiveness of this agent, while plasmid-associated factors are responsible for triggering arthritogenicity.

* Corresponding author. Mailing address: Servicio de Bacteriología, Unidad de Espiroquetas y Patógenos Especiales, Centro Nacional de Microbiología, 28220-Majadahonda, Madrid, Spain. Phone: (34) 91 509 7901. Fax: (34) 91 509 7966. E-mail: panda{at}isciii.es.

Editor: V. J. DiRita

Infection and Immunity, September 2002, p. 4851-4858, Vol. 70, No. 9
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.9.4851-4858.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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