Dose response effect of Paracoccidioides brasiliensis in an experimental model of arthritis

Abstract

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

Fig. 1. Radiological image of the right knee. A - A CGA animal on day 45 of the experiment presenting aspects compatible with normality. The arrow (a) indicates full epiphyseal line, the trabecular bone in the femur is regular (arrow (b)) and the regularity of the articular surface of the tibia and femur, indicated by the arrow (c). B - A GE animal (10⁵ yeast cells) on day 45 of the experiment displaying severe arthritis with joint destruction (arrow (a)), the absence of the patella-femoral joint space, shown by the arrow (b), and the absence of the epiphyseal line (arrow (c)). C - A GE animal on day 45 (inoculated with 10⁴ viable yeast cells) at the end of the study presents radiological findings consistent with mild arthritis. (a) Tibial subchondral cysts, (b) discontinuity of the articular surface of the tibia and (c) points to a deformity of the epiphyseal line adjacent to a subchondral cyst. D - A GE animal on day 15 (inoculated with 10³ viable yeast cells) presenting radiological findings consistent with mild arthritis. It is observed that the epiphyseal line is preserved (a) and that there are lytic lesions in the tibia and patella.

Fig. 2. Photomicrographs of histological longitudinal sections from the knees of the experimental animals. A - Articulation of a normal animal, GCA (HE 4x). B - Synovial membrane with swelling and granuloma from an animal after 15 days of exposure to a dose of 10³ yeast cells (HE 4x). C - Synovial swelling with a large amount of P. brasiliensis cells, indicated by the arrow, from an animal exposed to a dose of 10⁴ yeast cells for 15 days (Grocott 4x). D - Detail of figure (C) with a large amount of P. brasiliensis cells with budding (Grocott 40x). E - Pannus formation with the emergence of vessels (left arrow) and the multilayered synovial mesothelium (the arrows to the right) at 45 days exposure (HE 10x). F - Granuloma with a giant cell bone inside (HE 10x). G - Granuloma bone above the epiphyseal line, focusing on the necrosis inside, in one animal after 45 days of exposure to 10⁵ viable yeast cells (HE 4x). H - The arrow indicates the area of cartilage destruction, with a deep necrotic area after 45 days of exposure to 10⁵ viable yeast cells (HE 4x). I - Detail of figure (F) with a giant cell with P. brasiliensis cells inside (HE 100x).

Fig. 3. ELISA results for rat sera and gp43. The graph shows the mean optical density (1:50 dilution) obtained in the study groups after infection. * p = 0.0001 and p = 0.002 (ANOVA test) between the experimental groups at days 15 and 45.