Infection and inflammation in skeletal muscle from nonhuman primates infected with different genospecies of the Lyme disease spirochete Borrelia burgdorferi

Abstract

Lyme borreliosis is a multisystemic disease caused by various genospecies of the spirochete Borrelia burgdorferi. To investigate muscle involvement in the nonhuman primate (NHP) model of Lyme disease, 16 adult Macaca mulatta animals inoculated with strain N40 of B. burgdorferi sensu strictu by syringe or by tick bite or with strain Pbi of B. burgdorferi genospecies garinii by syringe were studied. Animals were necropsied while immunosuppressed on day 50 (two animals each inoculated with B. burgdorferi N40 by syringe and with B. garinii Pbi by syringe) or on day 90, 40 days after immunosuppression had been discontinued (four animals each inoculated with strain N40 by syringe, with strain N40 by tick bite, and with strain Pbi by syringe). Skeletal muscles removed at necropsy were studied by (i) microscopic examination of hematoxylin-eosin-stained sections for inflammation and tissue injury; (ii) immunohistochemical and digital image analyses for antibody and complement deposition and cellular inflammation; (iii) Western blot densitometry for the presence of antibodies; and (iv) reverse transcription-PCR for measurement of the spirochetal load or C1q (the first component of the complement cascade) synthesis. The results showed that N40 was more infectious for NHPs than Pbi. NHPs inoculated with N40 but not with Pbi developed myositis. The inflammation in skeletal muscle was more severe in NHPs inoculated with N40 by syringe than in those inoculated by tick bite. The predominant cells in the inflammatory infiltrate were T cells and plasma cells. The deposition of antibody and complement in inflamed muscles from N40-inoculated NHPs was significantly higher than that in Pbi-inoculated NHPs. The spirochetal load was very high in the two N40-inoculated NHPs examined while they were immunosuppressed but decreased to minimal levels in the NHPs when immunocompetence was restored. We conclude that myositis can be a prominent feature of Lyme borreliosis depending on the infecting organism and host immune status.

FIG. 1.

Immunosuppression of the majority of NHPs with dexamethasone to increase the probability of infection, starting 1 week prior to inoculation. Some NHPs were necropsied 40 days after discontinuation of dexamethasone (TISP; broken line). Others were necropsied while still receiving dexamethasone (IS; solid line).

FIG. 2.

B. burgdorferi-specific antibodies of the IgM (A) or IgG (B) isotype in necropsy sera from TISP NHPs 90 days after inoculation with B. burgdorferi sensu stricto strain N40 by needle (group 1) or by tick bite (group 2) or with B. garinii strain Pbi by needle (group 3). An ELISA was carried out with homologous sonicates and sera diluted 1:500. Data are reported as means and SDs.

FIG. 3.

Total specific antibody production in TISP NHPs 105 (×) and 321 (▪) after intradermal inoculation with B. garinii strain Pbi. An ELISA was carried out with homologous sonicates and sera diluted 1:200.

FIG. 4.

B. burgdorferi sensu stricto strain N40 in skeletal muscle from an IS NHP 50 days after intradermal inoculation. Arrowheads indicate spirochetes in the endomysium. Immunohistochemical analysis was carried out with hyperimmune rabbit serum. Magnifications, ×360 in the main panel and ×900 in the inset. Bar, 50 μm.

FIG. 5.

Myositis in a TISP NHP 90 days after needle inoculation with B. burgdorferi sensu stricto strain N40. (A) HE stain (magnification, ×400). (B) T-cell immunostain (CD3) (magnification, ×200). (C) Plasma cell immunostain (p63) (magnification, ×200). (D) Ki67 immunostain (magnification, ×200). (E) IgM immunostain (magnification, ×200). (F) IgG immunostain (magnification, ×200). Bar, 100 μm.

FIG. 6.

CD3 (A) or p63 (B) immunostaining in skeletal muscle from TISP NHPs inoculated with strain N40 by needle (group 1) or by tick bite (group 2) or with strain Pbi by needle (group 3). Results are given as mean and SD sum density in arbitrary units (CD3) or sum area in square micrometers per ×40 microscopic field (p63). (C) Immunostaining for various inflammatory markers in skeletal muscle from a strain N40 needle-inoculated TISP NHP (no. 199), shown in mean and SD square micrometers per ×40 microscopic field. Ki67, proliferation marker; CD3, T-cell marker; CD20, B-cell marker; Ham56, macrophage marker; p63, plasma cell marker.

FIG. 7.

Comparison of antibody (IgG and IgM) and complement (C1q) deposition in skeletal muscle from TISP NHPs inoculated with strain N40 by needle (group 1; ▥) or by tick bite (group 2; ▨) or with strain Pbi by needle (group 3; ▩). Results are given as mean and SD sum area in 10⁴ square micrometers per ×40 microscopic field.

FIG. 8.

Total antibodies of the IgM (A) or IgG (B) isotype in necropsy sera from TISP NHPs inoculated with strain N40 by needle (group 1) or by tick bite (group 2) or with strain Pbi by needle (group 3). Data are reported as mean and SD.