Nt manifestations and for post-treatment persistence of B. burgdorferi in mice. The results demonstrate that, indeed, the infection of mice with a B. burgdorferi strain that expresses both DbpA and B adhesins enables such progression of the infection that leads to arthritis development and post-treatment persistence. Results of our immunosuppression experiments suggest that the persisting material in the joints of mice infected with DbpA and B expressing bacteria and treated with ceftriaxone is DNA or DNA containing remnants rather than live bacteria.Materials and Methods B. burgdorferi strainsThe study was conducted using previously characterized B. burgdorferi strains [16]. dbpAB knock out strain, dbpAB/E22/1 (dbpAB), the DbpA and B expressing strain, dbpAB/ dbpAB/2 (dbpAB/dbpAB), the DbpA expressing strain, dbpAB/dbpA/1 (dbpAB/dbpA), and the DbpB expressing strain, dbpAB/dbpB/1 (dbpAB/dbpB) in B. burgdorferi B31 5A13 background are identical in all other aspects of their genetic composition but differ in the ability to express DbpA and/or B. The spirochetes were cultivated in Barbour-Stoenner-Kelly II (BSK II)PLOS ONE | DOI:10.1371/journal.pone.0121512 March 27,2 /DbpA and B Promote Arthritis and Post-Treatment Persistence in Micemedium containing kanamycin (200 g/ml, Sigma-Aldrich, St. Louis, MO, USA) and gentamycin (50 g/ml, Biological Industries, Beit-Haemek, Israel) at 33 . The minimal inhibitory concentration (MIC) of ceftriaxone was determined by culturing dbpAB/dbpAB and dbpAB in two-fold dilutions of the antibiotic in BSK II medium covering a concentration range of 0.5?.002 g/ml. Dark-field microscopy was used to detect the growth of the bacteria.Ethics StatementThis study was carried out in strict accordance with the recommendations in the Finnish Act on the Use of Animals for Experimental Purposes of Ministry of Agriculture and Forestry in Finland. The protocol was approved by the National Animal Experiment Board in Finland (permission number STH619A). All efforts were done to minimize suffering of the animals.Experimental designFour weeks old female C3H/HeNhsd (C3H/He) mice (Harlan, Netherlands) were infected with 106 dbpAB/dbpAB (40 mice), dbpAB/dbpA (8 mice), dbpAB/dbpB (8 mice) or dbpAB (38 mice) bacteria by intradermal syringe inoculation in the lower back. Twelve control animals were injected with an equal volume of PBS. In experiment I (Fig. 1), four animals were infected with dbpAB/dbpAB (group 2), eight with dbpAB/dbpA (group 3), eight with dbpAB/dbpB (group 4), and two with dbpAB (group 5). Two uninfected animals (group 1) were negative controls. The development of joint manifestations was monitored by measuring the medio-lateral diameter of the hind tibiotarsal joints once a week. The measurer was blinded to the group’s identity. The mice were killed at seven weeks of infection. Tissue samples from ear, bladder and hind tibiotarsal joint were collected for culture. In experiment II, 20 animals were infected with dbpAB/dbpAB (purchase (S)-(-)-Blebbistatin groups 7, 9 and 11) and 20 animals with dbpAB (groups 8, 10 and 12). Two uninfected animals (group 6) were negative controls. Sixteen animals (groups 9 and 10) were treated with ceftriaxone and 16 animals (groups 11 and 12) with ceftriaxone and anti-TNF-alpha. The ceftriaxone treatment was started at two weeks and the anti-TNF-alpha treatment at seven weeks of infection. Ceftriaxone (Rocephalin1, Roche, Mannheim, Germany) was CGP-57148B biological activity administered twice a day 25 mg/kg intraperitoneally for five days.Nt manifestations and for post-treatment persistence of B. burgdorferi in mice. The results demonstrate that, indeed, the infection of mice with a B. burgdorferi strain that expresses both DbpA and B adhesins enables such progression of the infection that leads to arthritis development and post-treatment persistence. Results of our immunosuppression experiments suggest that the persisting material in the joints of mice infected with DbpA and B expressing bacteria and treated with ceftriaxone is DNA or DNA containing remnants rather than live bacteria.Materials and Methods B. burgdorferi strainsThe study was conducted using previously characterized B. burgdorferi strains [16]. dbpAB knock out strain, dbpAB/E22/1 (dbpAB), the DbpA and B expressing strain, dbpAB/ dbpAB/2 (dbpAB/dbpAB), the DbpA expressing strain, dbpAB/dbpA/1 (dbpAB/dbpA), and the DbpB expressing strain, dbpAB/dbpB/1 (dbpAB/dbpB) in B. burgdorferi B31 5A13 background are identical in all other aspects of their genetic composition but differ in the ability to express DbpA and/or B. The spirochetes were cultivated in Barbour-Stoenner-Kelly II (BSK II)PLOS ONE | DOI:10.1371/journal.pone.0121512 March 27,2 /DbpA and B Promote Arthritis and Post-Treatment Persistence in Micemedium containing kanamycin (200 g/ml, Sigma-Aldrich, St. Louis, MO, USA) and gentamycin (50 g/ml, Biological Industries, Beit-Haemek, Israel) at 33 . The minimal inhibitory concentration (MIC) of ceftriaxone was determined by culturing dbpAB/dbpAB and dbpAB in two-fold dilutions of the antibiotic in BSK II medium covering a concentration range of 0.5?.002 g/ml. Dark-field microscopy was used to detect the growth of the bacteria.Ethics StatementThis study was carried out in strict accordance with the recommendations in the Finnish Act on the Use of Animals for Experimental Purposes of Ministry of Agriculture and Forestry in Finland. The protocol was approved by the National Animal Experiment Board in Finland (permission number STH619A). All efforts were done to minimize suffering of the animals.Experimental designFour weeks old female C3H/HeNhsd (C3H/He) mice (Harlan, Netherlands) were infected with 106 dbpAB/dbpAB (40 mice), dbpAB/dbpA (8 mice), dbpAB/dbpB (8 mice) or dbpAB (38 mice) bacteria by intradermal syringe inoculation in the lower back. Twelve control animals were injected with an equal volume of PBS. In experiment I (Fig. 1), four animals were infected with dbpAB/dbpAB (group 2), eight with dbpAB/dbpA (group 3), eight with dbpAB/dbpB (group 4), and two with dbpAB (group 5). Two uninfected animals (group 1) were negative controls. The development of joint manifestations was monitored by measuring the medio-lateral diameter of the hind tibiotarsal joints once a week. The measurer was blinded to the group’s identity. The mice were killed at seven weeks of infection. Tissue samples from ear, bladder and hind tibiotarsal joint were collected for culture. In experiment II, 20 animals were infected with dbpAB/dbpAB (groups 7, 9 and 11) and 20 animals with dbpAB (groups 8, 10 and 12). Two uninfected animals (group 6) were negative controls. Sixteen animals (groups 9 and 10) were treated with ceftriaxone and 16 animals (groups 11 and 12) with ceftriaxone and anti-TNF-alpha. The ceftriaxone treatment was started at two weeks and the anti-TNF-alpha treatment at seven weeks of infection. Ceftriaxone (Rocephalin1, Roche, Mannheim, Germany) was administered twice a day 25 mg/kg intraperitoneally for five days.
