An immunocompromised murine model of chronic Bartonella infection

Abstract

Bartonella are ubiquitous gram-negative pathogens that cause chronic blood stream infections in mammals. Two species most often responsible for human infection, B. henselae and B. quintana, cause prolonged febrile illness in immunocompetent hosts, known as cat scratch disease and trench fever, respectively. Fascinatingly, in immunocompromised hosts, these organisms also induce new blood vessel formation leading to the formation of angioproliferative tumors, a disease process named bacillary angiomatosis. In addition, they cause an endothelial-lined cystic disease in the liver known as bacillary peliosis. Unfortunately, there are as yet no completely satisfying small animal models for exploring these unique human pathologies, as neither species appears able to sustain infection in small animal models. Therefore, we investigated the potential use of other Bartonella species for their ability to recapitulate human pathologies in an immunodeficient murine host. Here, we demonstrate the ability of Bartonella taylorii to cause chronic infection in SCID/BEIGE mice. In this model, Bartonella grows in extracellular aggregates, embedded within collagen matrix, similar to previous observations in cat scratch disease, bacillary peliosis, and bacillary angiomatosis. Interestingly, despite overwhelming infection later in disease, evidence for significant intracellular replication in endothelial or other cell types was not evident. We believe that this new model will provide an important new tool for investigation of Bartonella-host interaction.

Figure 1

Bartonella taylorii causes sustained chronic blood stream infection in SCID/Beige mice. A: A/J, SCID, and SCID/Beige mice were inoculated by intraperitoneal injection with approximately 1.5 × 10¹⁰ bacterial genome equivalents of B. taylorii. At indicated time points after infection, blood was collected and plated on bacterial media to determine colony forming units (CFU) as shown. B: SCID/Beige mice were infected with B. taylorii LL-WM9 at approximately 3 × 10⁹ (H, 4 mice) or 1 × 10⁹ (L, 2 mice) genome copies. Blood was collected at indicated times postinfection and analyzed by qPCR to determine B. taylorii gltA gene copy number. As expected, the gltA sequence was not amplifiable from uninfected control animals (2 mice, data not shown).

Figure 2

Liver, renal, and splenic pathology four months postinfection. A: Liver demonstrating multiple lesions, low power. B: Liver lesion, higher power. Note slightly purple filamentous material (e.g., arrow) that corresponds to bacteria in Steiner-stained sections. C: Liver nonlesional area showing extramedullary hematopoiesis. Megakaryocyte (arrowhead), myeloid cells (arrows). D: Kidney lesion, higher power. E: Spleen from infected animal with obliteration of normal architecture. Inset: high power showing myeloid cells (arrows) and a megakaryocyte (arrowhead). F: Spleen from uninfected control, arrows show hemosiderin pigment, less prominent but still visible in infected spleens. Original magnifications of A–F and insets for E and F: ×40, ×200, ×500, ×200, ×40, ×40, and ×500X and ×500X, respectively.

Figure 3

Steiner stain identifies organisms within affected tissues. A: Large clusters of organisms (arrows) are found within the matrix of liver lesions. B: Liver sinusoid adjoining lesional area. Note organisms surrounding a vessel (arrowhead) and invading into the lumen of the sinusoid (arrow). C: Kidney with organisms surrounding and invading into the wall of a peritubular arteriole (arrow) and in interstitial tissue (arrowhead). D: Spleen demonstrating abundance of infecting organisms growing as large aggregates (arrows). Original magnifications of A–D: ×500, ×1000, ×1000, and ×500, respectively.

Figure 4

Trichrome stain demonstrates collagen-deposition response to bacteria. A: Liver showing blue-staining fibrillar collagen component of lesional area. B: Higher power of liver lesion showing relationship of collagen fibers (arrowheads) to acid fuchsin-staining bacteria (arrows). C: Kidney demonstrating collagen deposition in interstitial tissue in response to bacteria (not resolvable at this power). D: Spleen showing subcapsular collagen deposition in association with clusters (arrow) of acid-fuschin staining organisms. Original magnifications of A–D: ×200, ×1000, ×200, and ×500, respectively.

Figure 5

Ultrastructural pathology of the liver. A: Lesional area (with fibrillar matrix indicated by arrows). B: Fibers within lesions (arrow) show banding pattern (bracket) consistent with collagen. C: Stellate-shaped eukaryotic cells within lesion matrix show grossly dilated, rough endoplasmic reticulum (arrow) indicative of a high degree of synthetic activity. D: Higher power image of eukaryotic cell that appears to be laying down new collagen fibers (arrow) perpendicular to its surface. E: Bacteria within tissue show trilaminar staining pattern (arrow) characteristic of Gram-negative bacteria and a rough outer surface (arrowhead). F: Liver sinusoid with bacteria (arrow) tracking just outside of a sinusoidal space. Magnifications in A–F: ×1,700, ×26,900, ×6,400, ×10,100, ×33,700, and ×1,700, respectively.

Figure 6

Ultrastructural pathology of the spleen. A: Lower power image demonstrating myeloid infiltrate (arrowheads), bacteria (short arrows), red cells (asterisk), and edema (electron-lucent areas, long arrow). B: Higher power images showing bacteria surrounded by a relatively haphazard arrangement of collagen fibers (arrows) in comparison with liver lesions and edema (long arrow). C: Bacterial aggregates embedded in an osmophilic granular matrix (arrows). D: Higher power image of matrix (arrows) and embedded bacteria. Magnifications in A–D: ×1700, ×6,500, ×1,700, and ×17,100, respectively.