SCID Mouse Model for Lethal Q Fever

Mice.SCID (C.B-17/Icr-scid/scid) mice and immunocompetent C.B-17 (C.B-17/Icr-+/+) mice were obtained from Japan CLEA Inc. (Tokyo, Japan). A/J mice were obtained from Japan SLC Inc. (Shizuoka, Japan). Five- to 6-week-old female mice were used in the experiments. They were housed under sterile conditions at all times. All procedures were done under the guidelines for animal experiments at Gifu University.

Microorganism.The Nine Mile I strain of C. burnetii was maintained in mice by passage in spleen homogenates. The spleen homogenates were prepared in sucrose phosphate glutamate, kept at −80°C, and diluted with phosphate-buffered saline (PBS). C. burnetii in the homogenate was titrated to the 50% tissue culture infectious dose (TCID₅₀) in buffalo green monkey (BGM) cells by the indirect immunoperoxidase method (24).

Inoculation of mice and clinical studies.To compare the pathogenicities of C. burnetii in immunodeficient and immunocompetent mice, SCID mice (n = 11), C.B-17 mice (n = 6), and A/J mice (n = 6) were inoculated intraperitoneally with 10 TCID₅₀ of C. burnetii. As controls, SCID mice (n = 10), C.B-17 mice (n = 6), and A/J mice (n = 6) were mock inoculated with PBS. The mice were observed for 37 days, which is the time at which the last C. burnetii-infected mouse died. To compare the dose responses of the immunodeficient and immunocompetent mice, SCID mice (n = 6) were inoculated intraperitoneally with 0.5 ml of serial 10-fold dilutions (10⁴ to 10⁻⁵ TCID₅₀) of C. burnetii. C.B-17 mice (n = 6) were similarly inoculated with 10-fold dilutions (10⁴ to 10⁻³ TCID₅₀) of C. burnetii. As controls, SCID mice (n = 6) and C.B-17 mice (n = 6) were mock inoculated with PBS. The SCID mice were observed for 60 days, and the C.B-17 mice were observed for 30 days. The 50% lethal dose (LD₅₀) was calculated by the Behrens-Kärber method (5). Clinical signs and body weight were recorded daily. Relative body weight is the weight on a given day divided by the body weight on the day of inoculation. Blood samples were obtained by puncture of the heart under anesthesia before euthanasia. At necropsy, the spleen and liver were weighed, and a part of each organ was stored at −80°C. The rest of the spleen and liver and the heart, lungs, and kidneys were preserved in 10% formalin PBS.

Histopathology and immunocytochemistry.The organs of the mice that received 10 TCID₅₀ of C. burnetii and the control mice were examined. Sections of paraffin-embedded organs were prepared and stained with hematoxylin and eosin. The distribution of C. burnetii was examined by immunocytochemistry, using an anti-C. burnetii rabbit antiserum, goat anti-rabbit immunoglobulins (DAKO Japan, Kyoto, Japan), and avidin-biotin complex (ABC; Vector Laboratories, Burlingame, Calif.), as described elsewhere (4). The number of C. burnetii-positive cells in a section was scored as follows: none, −; a few cells in a separate part, +; several cells assembled in a specific part, ++; and cells throughout the section, +++.

Serology.Immunoglobulin G antibodies to phase I and II C. burnetii from C.B-17 and A/J mice were detected by an indirect immunofluorescence test (K. K. Htwe, K. Amano, Y. Sugiyama, K. Yagami, N. Minamoto, A. Hashimoto, T. Yamaguchi, H. Fukushi, and K. Hirai, Vet. Rec. 131:490, 1992) to determine whether the mice were infected. The serology of the SCID mice was not examined.

PCR.DNA was extracted from liver and spleen homogenates using a DNA extraction kit, SepaGene (Sanko Junyaku Co., Tokyo, Japan). The com1 gene fragment of C. burnetii was amplified by nested PCR using the primers OMP1-OMP2 and OMP3-OMP4 (30). To avoid false positives, DNA extraction and PCR were performed carefully according to guidelines described previously (14).

Statistical analysis.Differences between organ weights in control and infected mice were determined by Student's t test or Welch's t test following an F test. P values of <0.05 were regarded as significant.

Clinical signs and gross findings in SCID and immunocompetent mice.All the SCID mice infected with 10 TCID₅₀ of C. burnetii showed the same symptoms and died. The onset and duration of the clinical signs were as follows: 7 days postinoculation (p.i.), ruffled fur; 9 to 13 days p.i., hunchback appearance and inactivity; 27 to 31 days p.i., lethargy. The onset of a hunchback appearance and inactivity correlated with loss of body weight. Body weight continued to decline until death (Fig. 1). The survival period was 33 ± 2.5 days. None of the C.B-17 or A/J mice showed any clinical signs or died. The relative body weights of C.B-17 mice and A/J mice that were infected with 10 TCID₅₀ of C. burnetii were similar to those of the control mice.

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FIG. 1.

Weight changes of SCID mice inoculated with C. burnetii (solid circles) and PBS (open circles). A significant difference (P < 0.005) between infected and control mice was found 10 days p.i. Relative body weight is the body weight on a particular day divided by the body weight on the day of inoculation. Points represent means, error bars indicate standard deviations, and † indicates death of the mouse.

Hepatosplenomegaly was prominent in the SCID mice (Table 1), and the spleen had a pale color while the liver had necrotic foci. The A/J mice showed mild hepatosplenomegaly, whereas the C.B-17 mice showed only mild splenomegaly. In the C.B-17 and A/J mice, the liver did not have necrotic foci and the color of the spleen was normal.

None of the control mice showed any clinical signs or died, and no gross findings were detected.

Characteristic lesions with C. burnetii organisms in SCID mice.The lesions in SCID mice infected with 10 TCID₅₀ of C. burnetii were much more severe than the lesions in immunocompetent mice infected with 10 TCID₅₀ of C. burnetii (Table 2). The most characteristic and common feature observed in the lesions of SCID mice was severe cell infiltration. Almost all infiltrated cells were morphologically macrophages, and some were neutrophils. These macrophages were dilated, giving a vacuolated appearance, and were densely packed with basophilic granules, which varied from coarse to fine and were present in several extra- and intracellular forms. Immunocytochemistry revealed that these granules were C. burnetii organisms.

The hearts had severe infiltration of vacuolated macrophages in the epicardium and endocardium, including the valvular part. Focal calcification, evidence of chronic lesions, was observed in the epicardium and myocardium (Fig. 2A). Vacuolated macrophages were also found in the myocardium (Fig. 2B). In the kidney, the glomeruli were typically infiltrated with vacuolated macrophages (Fig. 2C), and the proximal tubules exhibited hyaline degeneration.

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FIG.2.

Heart (A and B) and kidney (C) sections from SCID mouse infected with C. burnetii. (A) Calcifications were observed in severely infiltrated lesions (arrows). There was macrophage infiltration in the epicardium (EP) and myocardium (MC). The macrophages were expanded and packed with granules (arrowheads). Hematoxylin and eosin staining; magnification, ×200. (B) C. burnetii antigens were detected as brown granules (arrowheads). ABC method; magnification, ×400. EN, endocardium; V, ventricle. (C) C. burnetii antigen-positive cells were characteristic in glomeruli (arrowheads). ABC method; magnification, ×200.

The lungs, spleen, and liver, which are the general target organs of C. burnetii in experimental animals, had severe lesions and many C. burnetii organisms. In the lungs, cells accumulated in intra-alveolar septi, stroma adjacent to airways, or venules (Fig. 3, top). The lumens contained no exudate. The spleen and liver did not retain their original structures due to bursting with macrophages and numerous vacuoles, which were probably the remains of necrotic cells. The margins of vacuoles were strongly immunopositive (Fig. 3, middle and bottom). C. burnetii organisms were also present within the hepatocytes (Fig. 3, middle). The liver also had some sporadic microabscesses, mainly containing macrophages, neutrophils, or lymphocytes. Additionally, the liver exhibited mild extramedullary hematopoiesis and contained a few megakaryocytes. Neither granulomas nor fibroblasts were found in any organs of the SCID mice.

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FIG.3.

Immunocytochemistry of the lung (top), liver (middle), and spleen (bottom) of SCID mouse infected with C. burnetii. C. burnetii antigens were detected as brown granules. (Top) C. burnetii antigen-positive cells infiltrated particularly in the stroma adjacent to a bronchiole (BR). AL, alveolus. (Middle) C. burnetii antigens were also found in hepatocytes (arrowheads). (Middle and bottom) The margins of vacuoles were immunopositive (arrows). ABC method; magnification, ×200.

The spleens of the control SCID mice, which originally lacked characteristic lymphoid follicles due to the absence of T and B lymphocytes, were somewhat smaller than those of the immunocompetent mice. No significant lesions were found in the control SCID mice.

General lesions in immunocompetent mice.The lesions of immunocompetent C.B-17 and A/J mice infected with 10 TCID₅₀ of C. burnetii were mild (Table 2) and were probably residual lesions, as reported elsewhere (3). The lesions of these two mouse strains were not noticeably different. A few small granulomas characterized by unexpanded macrophages, lymphocytes, or neutrophils were observed in the spleen, liver, and lungs. No lesions were observed in the heart or kidney. No significant lesions were found in control immunocompetent mice. Sections from both C. burnetii-inoculated and control immunocompetent mice were immunocytochemically negative and did not show nonspecific binding of ABC.

In the mice infected with 10 TCID₅₀ of C. burnetii, the antibody titers to phase I and II C. burnetii ranged from 1:256 to 1:1,024 and from 1:512 to 1:1,024, respectively, and were equivalent in the two mouse strains. The livers and spleens of the mice were PCR positive. No C. burnetii-specific antibodies or C. burnetii DNA was detected in the control mice.

Dose responses in SCID and immunocompetent mice.To determine the LD₅₀ and to observe the dose response in SCID mice, graded doses of C. burnetii (10⁴ to 10⁻⁵ TCID₅₀) were administered. All SCID mice that died in this experiment demonstrated clinical symptoms in the same progression and died within 60 days. The mice that died included all the ones that were given 10⁵ to 10⁻² TCID₅₀, four of six that were given 10⁻³ to 10⁻⁴ TCID₅₀, and one of six that were given 10⁻⁵ TCID₅₀ (Table 3). None of the diseased mice recovered. The LD₅₀ of C. burnetii in the SCID mouse was <10⁻⁴ TCID₅₀. Both the latent period (data not shown) and the survival period were inversely proportional to the inoculum size. Hepatosplenomegaly was observed in the dead SCID mice and was proportional to the survival period, but not to the inoculum size. Among the C.B-17 mice, only those receiving 10⁴ and 10³ TCID₅₀ showed ruffled fur, hunchback appearance, inactivity, and body weight loss, but they recovered within 15 days p.i. None of the C.B-17 mice died. Splenomegaly, which was proportional to the inoculum size, was observed in mice given 10⁴ to 10⁻³ TCID₅₀. Hepatomegaly was observed only in mice administered 10⁴ and 10³ TCID₅₀.