The hind tibiotarsal joints once a week. Each curve represents the mean of the study group. Asterisks denote significant difference from uninfected mice (P 0.05). Paraffin-embedded tissue sections were prepared from dbpAB/dbpAB (D; group 7) and dbpAB (E; group 8) infected and uninfected control (F; group 6) mice at 15 weeks of infection and stained for HE. Arrowheads indicate the synovial membrane, and asterisks indicate articular ICG-001 cancer cartilage surface in panels D and E. The original magnification in panels D and E is ?00 and in panel F ?0. Panel F indicates the anatomical structure shown in panels D and E. “Cef” Ceftriaxone treatment, “aT” anti-TNFalpha treatment. doi:10.1371/journal.pone.0121512.gSerology using the whole cell antigen and performed on post mortem serum samples indicated that all animals mounted a clear IgG response against both B. burgdorferi strains (Fig. 3A, groups 7 and 8). Antibodies against C6 peptide were significantly increased only in the samples of dbpAB/dbpAB infected mice (S1A Fig., groups 7 and 8). dbpAB/dbpAB infected micePLOS ONE | DOI:10.1371/journal.pone.0121512 March 27,7 /DbpA and B Promote Arthritis and Post-Treatment Persistence in MiceTable 1. B. burgdorferi culture results of Experiment I at seven weeks of infection. Culture at 7 wk Group 1 2 3 4 5 Strain/treatment Uninfected dbpAB/dbpAB dbpAB/dbpA dbpAB/dbpB dbpAB Ear 0/2 4/4 8/8 5/8 0/2 Bladder 0/2 4/4 7/7a 8/8 2/2 Joint 0/2 4/4 7/8 8/8 2/a One sample not available for culture doi:10.1371/journal.pone.0121512.tdeveloped significantly elevated antibody levels also against DbpA and B (S1B and C Fig., group 7). B. burgdorferi culture of post mortem tissue samples demonstrated widespread infection with positive culture results in all analysed tissues of dbpAB/dbpAB infected mice and in the tissues of three out of four dbpAB infected animals (Table 2, groups 7 and 8). SP600125 web Amplification of B. burgdorferi flaB and ospA genes using DNA extracted from joint tissue samples of dbpAB/dbpAB infected mice corroborated the presence of B. burgdorferi in the joints of all dbpAB/dbpAB infected and untreated mice (Table 3, group 7). Sensitivity of PCR appeared to be lower than the sensitivity of culture since 75 (? of ear samples of dbpAB/dbpAB infected mice that were all culture positive were positive in PCR. This was even more pronounced with the samples of dbpAB infected animals with only one out of three culture positive ear samples and none of the three culture positive bladder samples were PCR positive (Table 3, group 8). The results of the quantitative ospA PCR showed that there was no statistically significant difference in the bacterial load between the B. burgdorferi PCR positive joint samples of dbpAB/dbpAB and dbpAB infected mice (Fig. 4, groups 7 and 8). These results demonstrate that also the dbpAB strain is able to persistently infect mice, and that the bacterial load in the joints of dbpAB infected mice does not differ from the load of dbpAB/dbpAB infected mice at 15 weeks of infection.Effect of ceftriaxone and anti-TNF-alpha treatments on dissemination and arthritis developmentCeftriaxone was administered twice a day for five days during the third week of infection in subgroups of dbpAB/dbpAB and dbpAB infected mice (Fig. 1, groups 9?2). AntibioticTable 2. B. burgdorferi culture results of Experiment II at 6, 9 and 15 weeks of infection. Ear Culture Group 6 7 8 9 10 11 12 Strain/treatment Uninfected dbpAB/dbpAB dbpAB dbpAB/dbpAB + Cef2 dbpAB + Cef2 dbpA.The hind tibiotarsal joints once a week. Each curve represents the mean of the study group. Asterisks denote significant difference from uninfected mice (P 0.05). Paraffin-embedded tissue sections were prepared from dbpAB/dbpAB (D; group 7) and dbpAB (E; group 8) infected and uninfected control (F; group 6) mice at 15 weeks of infection and stained for HE. Arrowheads indicate the synovial membrane, and asterisks indicate articular cartilage surface in panels D and E. The original magnification in panels D and E is ?00 and in panel F ?0. Panel F indicates the anatomical structure shown in panels D and E. “Cef” Ceftriaxone treatment, “aT” anti-TNFalpha treatment. doi:10.1371/journal.pone.0121512.gSerology using the whole cell antigen and performed on post mortem serum samples indicated that all animals mounted a clear IgG response against both B. burgdorferi strains (Fig. 3A, groups 7 and 8). Antibodies against C6 peptide were significantly increased only in the samples of dbpAB/dbpAB infected mice (S1A Fig., groups 7 and 8). dbpAB/dbpAB infected micePLOS ONE | DOI:10.1371/journal.pone.0121512 March 27,7 /DbpA and B Promote Arthritis and Post-Treatment Persistence in MiceTable 1. B. burgdorferi culture results of Experiment I at seven weeks of infection. Culture at 7 wk Group 1 2 3 4 5 Strain/treatment Uninfected dbpAB/dbpAB dbpAB/dbpA dbpAB/dbpB dbpAB Ear 0/2 4/4 8/8 5/8 0/2 Bladder 0/2 4/4 7/7a 8/8 2/2 Joint 0/2 4/4 7/8 8/8 2/a One sample not available for culture doi:10.1371/journal.pone.0121512.tdeveloped significantly elevated antibody levels also against DbpA and B (S1B and C Fig., group 7). B. burgdorferi culture of post mortem tissue samples demonstrated widespread infection with positive culture results in all analysed tissues of dbpAB/dbpAB infected mice and in the tissues of three out of four dbpAB infected animals (Table 2, groups 7 and 8). Amplification of B. burgdorferi flaB and ospA genes using DNA extracted from joint tissue samples of dbpAB/dbpAB infected mice corroborated the presence of B. burgdorferi in the joints of all dbpAB/dbpAB infected and untreated mice (Table 3, group 7). Sensitivity of PCR appeared to be lower than the sensitivity of culture since 75 (? of ear samples of dbpAB/dbpAB infected mice that were all culture positive were positive in PCR. This was even more pronounced with the samples of dbpAB infected animals with only one out of three culture positive ear samples and none of the three culture positive bladder samples were PCR positive (Table 3, group 8). The results of the quantitative ospA PCR showed that there was no statistically significant difference in the bacterial load between the B. burgdorferi PCR positive joint samples of dbpAB/dbpAB and dbpAB infected mice (Fig. 4, groups 7 and 8). These results demonstrate that also the dbpAB strain is able to persistently infect mice, and that the bacterial load in the joints of dbpAB infected mice does not differ from the load of dbpAB/dbpAB infected mice at 15 weeks of infection.Effect of ceftriaxone and anti-TNF-alpha treatments on dissemination and arthritis developmentCeftriaxone was administered twice a day for five days during the third week of infection in subgroups of dbpAB/dbpAB and dbpAB infected mice (Fig. 1, groups 9?2). AntibioticTable 2. B. burgdorferi culture results of Experiment II at 6, 9 and 15 weeks of infection. Ear Culture Group 6 7 8 9 10 11 12 Strain/treatment Uninfected dbpAB/dbpAB dbpAB dbpAB/dbpAB + Cef2 dbpAB + Cef2 dbpA.
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