Conceived and designed the experiments: DS HF HE. Performed the experiments: DS MZ RL FF RR DL EH DB DG HE. Analyzed the data: DS MZ DB DG HF HE. Wrote the paper: DS HF HE.
The authors have declared that no competing interests exist.
Hantavirus pulmonary syndrome (HPS), also referred to as hantavirus cardiopulmonary syndrome (HCPS), is a rare but frequently fatal disease caused by New World hantaviruses. In humans HPS is associated with severe pulmonary edema and cardiogenic shock; however, the pathogenesis of this disease remains unclear largely due to a lack of suitable animal models for the study of disease progression. In this study we monitored clinical, virological, pathophysiological parameters and host immunological responses to decipher pathological factors and events in the lethal Syrian hamster model of HPS following intranasal inoculation of Andes virus. Transcriptional profiling of the host gene responses demonstrated a suppression of innate immune responses in most organs analyzed during the early stage of infection, except for in the lung which had low level activation of several pro-inflammatory genes. During this phase Andes virus established a systemic infection in hamsters, with viral antigen readily detectable in the endothelium of the majority of tissues analyzed by 7–8 days post-inoculation. Despite wide-spread infection, histological analysis confirmed pathological abnormalities were almost exclusively found in the lungs. Immediately preceding clinical signs of disease, intense activation of pro-inflammatory and Th1/Th2 responses were observed in the lungs as well as the heart, but not in peripheral organs, suggesting that localized immune-modulations by infection is paramount to pathogenesis. Throughout the course of infection a strong suppression of regulatory T-cell responses was noted and is hypothesized to be the basis of the aberrant immune activations. The unique and comprehensive monitoring of host immune responses to hantavirus infection increases our understanding of the immuno-pathogenesis of HPS and will facilitate the development of treatment strategies targeting deleterious host immunological responses.
New World hantaviruses, including Andes virus (ANDV), are rodent-borne pathogens which are associated with hantavirus pulmonary syndrome (HPS) and case fatality rates up to 50%. The pathogenesis of HPS remains unclear; however, it is believed to involve a delicate balance of virus infection and deleterious immune-modulations. In this study, exploiting pathological and immunological approaches, we investigate the disease mechanisms of HPS caused by ANDV in the lethal Syrian hamster model. Despite systemic viral replication, pathological findings were almost exclusively observed in the lungs of infected hamsters. The most striking observations came from monitoring the host responses in infected and control hamsters. During early infection, innate responses in infected hamsters were down-regulated in all organs except lung, which demonstrated slight increases in pro-inflammatory transcripts. Immediately preceding the symptomatic phase, considerable increases in pro-inflammatory and T-cell activating cytokine responses were observed in lung and heart tissue, but not in other organs. Interestingly, expression of regulatory T-cell marker genes that might control deleterious inflammatory responses were suppressed throughout infection, suggesting a role in the immune-dysregulation during hantavirus infection. The results of this study provide an increased understanding of HPS pathogenesis and will aid in the development of novel therapeutic strategies to counter HPS.
Hantaviruses (family
Clinically, HPS is defined as a febrile illness distinguished by diffuse, bilateral interstitial pulmonary infiltrates and compromised respiratory function which requires supplemental oxygen
The pathogenesis of HPS remains unclear but likely involves a versatile balance of viral replication and immune modulation resulting in increased vascular permeability.
Currently, only two disease models for hantaviruses have been described; infection of cynomolgus macaques (
Here we examined HPS disease progression in Syrian hamsters following intranasal inoculation of a lethal dose of ANDV. In the present study we show that ANDV down regulates the host innate immune response early during infection allowing systemic spread of the virus. In addition, we found that immediately preceding clinical onset of disease, activation of innate and Th1/Th2 responses were observed, primarily in lung and heart, while T-regulatory cell responses were largely absent. Our results indicate that tissue specific ANDV immune-modulation is a key factor in HPS pathogenesis.
Inoculation of hamsters with 200 FFU of ANDV via the intranasal route resulted in 100% lethality occurring between 10 and 13 days post infection (p.i.). Following challenge, hamsters appeared normal with no obvious signs of adverse effects caused by the inoculation procedures. Overt signs of disease were not apparent in any animals until day 9 p.i., with individual hamsters succumbing to infection approximately 12–36 hours post onset of clinical signs. The majority of terminally ill hamsters presented with lethargy, hunched posture and dyspnea, while some also showed cyanosis and/or epistaxis.
Radiographic imaging was utilized to assess respiratory disease progression at regular intervals during the course of infection in a subset of 5 animals, as well as at the time of necropsy for all hamsters. Changes were first observed between days 7–9 p.i. with a minority of hamsters showing mild lung infiltration. After day 9 p.i., notable increases in opacity were observed throughout the lungs of most infected animals. At the time of clinical disease onset (days 9–11 p.i.), moderate to severe pleural effusions with diffuse pulmonary infiltrates were visible, followed by rapid pulmonary consolidation with the most drastic changes observed within the last 48 hours preceding death (
Hamsters were infected with 200 FFU of Andes virus as outlined in the
Whole lungs were removed from Andes virus infected hamsters at indicated time points post-infection and lung to body weight ratios were determined. Individual dots represent data from a single hamster (expressed as a %) and are color coded to reflect the signs of infection/disease (as determined by clinical presentation and gross pathology of lungs, see inset for legend) of the specific animal. The blue dashed line represents the average lung to body weight ratio of control (uninfected) hamsters (n = 13) and the red dashed line represents the average ratios of infected hamsters at the indicated time point. Error bars represent the standard error of the mean. * Time point p<0.05; ** Time point p<0.01 (infected hamsters compared to controls).
Total RNA was extracted from tissue samples and tested for the presence of ANDV RNA as previously described
ANDV RNA was quantified in organs collected from infected hamsters at the indicated time points using primers and probe designed against the nucleoprotein coding region (A). Heart samples were not available for analysis at the day 10 time point due to insufficient levels of RNA. Error bars represent the standard error of the mean. LN, lymph nodes. Infectious ANDV titers were determined in lung samples from infected hamsters at the indicated time points using a focus-forming unit assay (B). Error bars represent the standard error of the mean.
Immunohistochemistry (IHC) was accomplished using a monoclonal antibody generated against the ANDV nucleoprotein. Viral antigen was first observed at day 7 p.i. in multiple organs and cell types including, the lung vascular endothelium and alveolar septal lining cells (
Hamsters were infected with 200 FFU of ANDV as outlined in the
Representative samples of heart, kidney, liver, lymph nodes and spleen sections collected at day 12 post-infection from a terminally ill, ANDV infected hamster are shown (H&E 200X, anti-Andes virus nucleoprotein IHC 400X). Extensive viral antigen was noted in endothelial cells of all tissues analyzed (black arrow), including endocardial endothelium in heart sections (open arrow) and glomerular tuft endothelium in kidney sections (circled areas) as well as hepatocytes and Kupffer cells in liver sections (denoted with an H and K, respectively) and dendritic cells and macrophages in lymph nodes (circled area). Despite the high frequency of antigen positivity in peripheral tissue samples, histological abnormalities were infrequent and largely unremarkable in these organs.
At the conclusion of each necropsy, hamsters were decapitated and the head was skinned, fixed in 10% neutral buffered formalin, decalcified and sectioned for histological analysis of the upper respiratory tract. Throughout the course of the study no discernable virus induced pathology was noted in the upper respiratory tract of infected hamsters. (A, 40X magnification of upper respiratory tract of infected hamsters collected at day 10 post-infection). Immunohistochemistry demonstrates the presence of ANDV infected olfactory epithelial cells (black arrow) and endothelium (open arrow) in the upper respiratory tract of hamsters at days 10 (B, 200X magnification) and 12 (C, 400X magnification) p.i.
Gross pathological abnormalities were consistently noted in the lungs of infected animals. Beginning at day 7 p.i., macroscopic lesions were observed in the lungs of 3 of 6 infected animals. The frequency and severity of the affected areas increased until the terminal stage of disease at which point lesions covered ≥80% of all lung lobes and were hemorrhagic in appearance (
Similar to the gross pathology observed at necropsy, microscopic abnormalities were predominantly observed in the lungs of infected hamsters and consisted of interstitial pneumonitis and perivascular pulmonary inflammation with increased numbers of macrophages and neutrophils, expanding interstitial spaces, alveolar and perivascular edema, hemorrhage and fibrin deposition (
At no time point were any histological abnormalities observed in the heart, spleen, kidney (
Pronounced leukocytosis, mainly consisting of neutrophilia and lymphocytosis, was observed in infected hamsters as early as day 3 p.i. and continuing throughout the terminal stages of disease (data not shown). Clinical chemistries were mostly unremarkable in infected hamsters and differences in platelet counts were not observed. A slight (average of 5%) increase in hematocrit was documented, though only at days 11–12 p.i.. Examination of coagulation parameters (aPTT, PT, TT, Protein S, Protein C and fibrinogen concentrations) demonstrated a moderate increase in PT and aPTT and decrease in plasma fibrinogen levels which corresponded to the clinical phase of disease in hamsters (
Group | aPTT (s, ±SEM) | PT (s, ±SEM) | Fibrinogen (mg/dL, ±SEM) | Thrombin time (s, ±SEM) | Protein C |
Protein S |
Control | 21.07±0.44 | 10.80±0.45 | 313.11±12.14 | 30.47±1.02 | 100 | 100 |
Day 1 | 20.05±0.99 | 9.75±0.15 | 347.13±22.74 | 26.65±0.68 | 100 | 100 |
Day 3 | 21.13±0.57 | 9.53±0.55 | 320.14±29.80 | 25.08±1.25 | 100 | 61.1 |
Day 5 | 21.55±0.20 | 9.43±0.44 | 265.20±37.56 | 27.92±1.26 | 92 | 74.4 |
Day 7 | 22.65±0.42 | 10.62±0.14 | 333.32±18.13 | 28.53±1.09 | 100 | 81.1 |
Day 8 | 22.02±1.18 | 11.50±0.18 | 324.52±10.88 | 26.42±1.42 | 100 | 91.1 |
Day 9 | 23.35±1.10 | 10.01±0.14 | 333.82±13.04 | 29.37±1.28 | 100 | 86.1 |
Day 10 | 24.98±1.53 | 12.14±1.36 | 290.33±59.70 | 32.57±3.87 | 100 | 91.7 |
Day 11 | 29.84±1.42 |
10.66±0.49 | 262.72±46.97 | 33.53±4.73 | 100 | 60.6 |
Day 12 | 43.10±3.46 |
13.70±2.07 |
148.90±47.02 |
24.08±3.44 | 96 | 59.6 |
p value | <0.001 | 0.0234 | 0.0115 | - | - | 0.0416 |
*Protein C and Protein S expressed as percent activity compared with control, uninfected hamsters.
Host responses (including IL- 1β, 2, 4, 6, 10, 12p35 and 21, TNF α, IFN γ, TGF β, STAT1 and 2, IRF1 and 2, Mx2 and FoxP3) were monitored in lung, heart, spleen, cervical lymph nodes and blood samples from infected hamsters and compared to levels of control animals using recently developed, hamster specific real-time qRT-PCR assays (
Host responses to ANDV infection were monitored using recently developed, hamster specific, real-time RT-PCR assays. The average fold changes in lung, heart, spleen, cervical lymph nodes and blood samples from a minimum of 6 infected animals per time point are shown. Data collected is normalized uninfected animals. Day 10 heart samples were not tested due to insufficient RNA concentrations following extraction. Spleen samples were not tested for STAT-2 or IRF-2 for the same reason.
Due to our small sampling size and the variability associated with differing responses to infection in outbred animals, a thorough statistical analysis of the temporal changes in cytokine transcripts within and between organs could not be accomplished. However, analyzing patterns of prolonged activation (i.e., ≥2 consecutive time points) revealed a significant amount of pro-inflammatory transcripts up-regulated in lungs during early infection (between days 1 and 7 p.i.), when compared to other organs examined (6 of 7 in lung, 0 of 7 for cervical lymph nodes and spleen each, p = 0.0210). At later time points, this trend continued in lung samples with 5 of 7 pro-inflammatory markers up-regulated compared 1 and 0 in spleen and lymph nodes respectively. Interestingly, during later disease (between days 7 and 12) a significant increase in the number of activated pro-inflammatory markers was observed in the heart when compared to lymph nodes and spleen (7 of 7 versus 0 and 1 respectively, p<0.01).
Host responses were also monitored in plasma samples from a subset of infected and control hamsters using a multiplex antigen capture-based assay as previously described
Plasma samples from control (n = 6) and Andes virus infected (n = 22) hamsters were tested for analytes using the RodentMAP version 2.0 platform. Samples from infected hamsters were divided into early (n = 6, collected on day 1 post-infection, p.i.), middle (n = 8, collected on days 7–8 p.i.), or late (n = 8, collected on days 11–12 p.i.) stages of disease. Shown are data from biomarkers that sufficiently cross-reactive with the mouse/rat specific assay. IP, inducible protein; M-CSF, macrophage-colony stimulating factor; MCP, monocyte chemoattractant protein; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor; VEGF, vascular endothelial cell growth factor; MDC, macrophage-derived chemokine; SCF, stem cell factor. * p<0.01, ** p<0.001; *** p<0.0001 (indicated time point to uninfected controls).
The pathogenesis of HPS has basically evolved from studying a limited number of surviving and lethal HPS cases
Intranasal ANDV inoculation established a systemic infection in hamsters with viral specific RNA detectable in blood and all tissues analyzed from day 7 p.i. on (
Previously, it has been reported that ANDV is capable of establishing a productive infection in primary respiratory epithelial cell cultures
The earliest signs of infection were manifest in hematology with pronounced increases of neutrophils and lymphocytes beginning at day 3 p.i. and continuing throughout the course of infection. Similar hematological changes have been described in HPS cases
Recently it was shown that ANDV infection of primary lung endothelial cells resulted in upregulation of VEGF and downregulation of vascular endothelial (VE)-cadherin, both of which are associated with increased vascular permeability
As known from other virulent RNA virus infections
It is well established that downregulation of Treg cells results in disrupted immune homeostasis leading to immunopathology, while a vigorous Treg response can result in viral persistence
Pathology was focused in the lungs of ANDV infected hamsters which correlates well with the early and eventually strong immune response to infection in the lung and the primarily respiratory disease manifestation. Other organs are clearly infected, with evidence for ANDV replication in endothelial cells, but with the exception of the heart, measurable Th1/Th2 immune responses could not be detected in these organs (
Our present study strongly suggests that HPS can be categorized as a severe disease induced by organ-specific pathological changes associated with dysregulation of organ-specific host responses. Supporting this, a study by Mori and colleagues demonstrated an increase of cytokine producing lung cells in HPS cases, but not other organs
The role of TNF α in the pathogenesis of HPS and particularly for increased vascular permeability is controversial
Of note is the increased IL-21 expression in heart tissue of ANDV infected hamsters. IL-21 plays a critical role in the differentiation of Th17 cells, a newly recognized subset of T cells with opposite function to Treg cells and involvement in autoimmune processes
The dogma of T cell mediated immunopathogenesis of hantavirus infections was recently challenged by Hammerbeck and Hooper
The timing of the increases in activated T cells noted by Hammerbeck and Hooper in undepleted hamsters appears to correlate with the onset of the aberrant immune responses observed in our study (
Attempts to quantify plasma cytokine responses in hamsters using reagents developed for other rodent species were largely unproductive. Most likely this is due to a lack of cross-reaction, which has been shown previously in Pichinde infected hamsters
In conclusion, following intranasal infection, a broad suppression of innate responses allows ANDV to establish an infection in the lower respiratory tract with subsequent systemic spread. Early measurable signs of infection are hematological abnormalities and pulmonary infiltration, primarily consisting of neutrophils and macrophages early, and macrophages and lymphocytes later in disease. A crucial time during infection lies around day 7 p.i., when initial immune suppression switches to immune activation which appears to be triggered through continuous suppression of host regulatory responses (i.e., Tregs), and uncontrolled viral replication. The terminal stage starts with the appearance of clinical signs around day 9 p.i. when radiographic changes can be observed. This period is short and animals succumb to infection within 36–48 hours, as has been described for human HPS cases. Virus dissemination seems to occur through viremia but the primary organ for virus replication and immune-associated damage through host responses is the lung followed by the heart. Thus, pathogenesis seems to be a result of a combination of virus replication with an early suppression of the host innate immune responses followed by a virus-triggered organ-specific deleterious host immune response resulting in pulmonary damage and cardiac suppression leading to respiratory distress.
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories (ASP #2009-43), and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in an AAALAC approved facility.
All work with infected hamsters and potentially infectious materials derived from hamsters was conducted in the Biosafety Level 4 facility at the Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Sample inactivation and removal was performed according to standard operating protocols approved by the local Institutional Biosafety Committee.
ANDV, strain Chile 9717869
At 12 hours and 1, 3, 5, 7, 8, 9, 10, 11 and 12 days p.i., one control and six infected hamsters were anesthetized via inhalational isoflurane and chest radiographs (ventral dorsal, right and left lateral) were taken using a portable digital radiography unit with a flat panel digital detector (TruDR, Sound-Eklin, Carlsbad, CA) and veterinary specific software (VET-PACS, London, United Kingdom). Hamsters were weighed, bled (EDTA and heparin treated 1.8 mL vacutainer tubes) and exsanguinated via cardiac puncture. Necropsies were performed to collect trachea, lung, liver, spleen, heart, kidney, digestive tract (stomach and intestines) and cervical lymph nodes. Lungs were removed, photographed, weighed, and a representative sample was taken from each lobe for histopathology (see below). In addition, a piece of the left lung was processed for RT-PCR as outlined below. Organ samples were processed for molecular analysis, histopathology and IHC. For molecular analysis, pieces of tissue (approximately 100 mg) were immersed in 1 mL of RNAlater (Qiagen, Valencia, CA) for overnight at 4°C, removed and stored at −80°C until RNA extraction. The remainders of each organ were fixed in 10% neutral buffered formalin for seven days at 4°C and processed as outlined below for histopathology and IHC. The head was skinned and fixed in 10% neutral buffered formalin for histological analysis of the upper respiratory tract. Lung weight to body weight ratios were calculated and pairwise comparisons of data from control and infected hamsters performed at each interval using t-tests.
Hamsters were assigned a clinical score based on signs of infection/disease as determined by clinical presentation and gross pathology of lungs as follows: no signs = indistinguishable from controls; mild = hamsters appeared lethargic, lesions on single lung lobes; moderate = onset of breathing abnormalities (rapid shallow breathes), lesions apparent on multiple lung lobes; severe = breathing distress, multi-focal lesions of increased size on all lung lobes; terminal = moribund, epistasis, lungs hemorrhagic in appearance.
Disease progression was monitored in a subset of five ANDV infected hamsters with chest radiographs taken at one to two day intervals until day 7 p.i. and twice daily (approximately 12 hours apart) from day 7 p.i. until the point of euthanasia.
Blood chemistry was monitored in heparinized blood using a portable iSTAT instrument (Abbott Point of Care, Princeton, NJ) with EC8+ cartridges measuring Na, K, Cl, TCO2, anion gap, glucose, urea nitrogen, hematocrit, hemoglobin, pH, pCO2, pO2, TCO2, HCO3, and base excess. Hematology, including white blood cell count, lymphocyte, platelet, reticulocyte and red blood cell counts, hematocrit values, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentrations, was performed on EDTA blood using a Hemavet 950FS (Drew Scientific, Waterbury, CT).
Due to the volume of plasma required for the coagulation examination, a second group of one control and six infected hamsters were exsanguinated via cardiac puncture at days 1, 3, 5, 7, 8, 9, 10, 11 and 12 p.i. with blood collected into 1.8 mL citrate vacutainers. Plasma was tested for coagulation parameters, including activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen concentration, and Protein S and Protein C activity, on a STart4 instrument using the PTT Automate, STA Neoplastine CI plus, STA Thrombin, Fibri-Prest automate, STA Staclot Protein S and Protein C kits, respectively (all from Diagnostica Stago, Parsippany, NJ). Data was analyzed using a one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparison post-test comparing values from infected hamsters to uninfected controls.
Formalin fixed tissues were processed and embedded in paraffin according to standard procedures. For sectioning of the nasal tract, the mandible and any extraneous muscle was removed from formalin fixed skulls to expose the cranium. Skulls were rinsed in running tap water, placed in a decalcifying solution consisting of 20% EDTA in sucrose (Newcomer Supply, Middleton WI) and allowed to sit at room temperature for 3–5 weeks, with 2–3 changes of decalcification solution over that period. Following decalcification, skulls were processed and embedded in paraffin.
Thin (5 µm) sections were cut and stained with hematoxylin and eosin or tested for the presence of viral antigen by IHC using a monoclonal antibody generated against the ANDV nucleoprotein (1:500 dilution, clone 1A8F6, Austral, San Ramon, CA). IHC was accomplished on a Discovery XT instrument (Ventana Medical Systems, Tucson, AZ) using a biotinylated goat anti-mouse (1:250 dilution, BioGenex, San Ramon, CA) secondary antibody and DAB Map kit. Following immunological staining, slides were counterstained with hematoxylin, dehydrated, cleared in xylene, and coverslipped. In addition, lung sections were stained for vascular endothelial growth factor (VEGF, 1:100 dilution, sc-507, Santa Cruz Biotechnology, Santa Cruz, CA) using a cross-reactive polyclonal antibody generated in rabbits essentially as outlined above with an alkaline phosphatase conjugated secondary antibody (Ventana Medical Systems).
Slides were evaluated by a Veterinary Pathologist. The scoring for H and E stained lung specimens were as follows: 1 = Minimally increased numbers of inflammatory cells within alveolar septae without widening of the septae; 2 = Mildly increased numbers of inflammatory cells within septae and mild expansion, or thickening, of septal walls and occasional extension of inflammation into the lumen of alveoli and bronchioles; 3 = Interstitial inflammation (alveolar septae and perivascular, peribronchiolar and peribronchial) and inflammation within alveoli and larger airways (inflammatory cells, fibrin, hemorrhage). Inflammation may be extensive and involve large areas of the tissue section; 4 = Changes are as described for #3; however, extent of lesions is greater and involves most of the tissue section.
RNA was extracted from solid tissues (30 mg pieces) and blood samples using RNeasy or QIAamp viral RNA kits (Qiagen), respectively, according to manufacturers' instructions. Immediately after extraction, RNA was quantified on a nanodrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE). Sample concentration was adjusted to 40 ng/µl and aliquots frozen at -80°C. Real-time quantitative (q) RT-PCR was performed on a rotor-gene 6000 instrument (Corbett Life Science, Sydney Australia) using QuantiFast probe reagents (Qiagen) and 200 ng of template RNA. The presence of ANDV RNA was quantified using a previously described nucleoprotein specific assay
Plasma samples from a subset of infected (n = 22) and control (n = 6) hamsters were sent to Rules-Based Medicine, Inc (Austin, TX) for analysis of cytokine and chemokine concentrations using a 58-biomarker Multi-Analyte Profile (MAP) approach (RodentMAP version 2.0). Samples from infected animals were divided into three categories; early (n = 6, collected on day 1 p.i.), middle (n = 8, collected on days 7–8 p.i.), and late (n = 8, collected on days 11–12 p.i.) stages of disease. Although this platform has only been validated against mouse and rat samples, recently it was suggested to work for selected hamster biomarkers, especially chemokines
Infectious viral loads were measured in a subset of lung homogenates (10% [w/v]) from infected hamsters using a focus forming unit assay essentially as previously described
GenBank accession numbers are as follows: IL-1β AB028497; IL-2 EU729351; IL-4 AF046213; IL-6 AB028635; IL-10 AF046210; IL-12p35 AB085791; IL-21 FJ664142; TNF α AF315292; IFN γ AF034482; TGF β AF046214; FoxP3 FJ 664148; Mx2 EU616539; IRF1 DQ092344; IRF2 AY714581; STAT1 DQ092343; STAT2 AB177399; ribosomal protein L18 DQ403027.
Host responses to Andes virus infection. Host responses to Andes virus infection were monitored using recently developed, hamster specific, real-time RT-PCR assays. Shown is the qRT-PCR data used to generate the heat maps in
(TIFF)
The authors thank Dr. John Mattoon (Department of Veterinary Clinical Sciences, Washington State University) for providing comments on the radiographs, Sandy Skorupa, Kathleen Meuchel and Lisa Kercher (Rocky Mountain Veterinary Branch, Division of Intramural Research, NIAID, NIH) for assistance in animal care and basic animal procedures and Anita Mora and Austin Athman (Division of Intramural Research, NIAID, NIH) for helping prepare the figures.