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Comp Med. Jun 2009; 59(3): 257–265.
Published online Jun 2009.
PMCID: PMC2733296

The Physiologic Responses of Dutch Belted Rabbits Infected with Inhalational Anthrax

Abstract

Bacillus anthracis, the causative agent of anthrax, is a category A priority pathogen that causes extensive damage in humans. For this reason, B. anthracis has been the focus of numerous studies using various animal models. In this study, we explored physiologic parameters in Dutch belted rabbits with inhalation anthrax to characterize the disease progression in this model. To this end, we infected Dutch belted rabbits with 100 LD50 B. anthracis Ames spores by nasal instillation and continuously recorded various physiologic parameters by using telemetry. In addition, samples were collected at selected times for serum chemistry, hematology, and blood gas analysis. The animals exhibited hemodynamic and respiratory changes that coincided with those reported in human cases of inhalational anthrax infection, including hypotension, altered heart rate, and respiratory distress. Likewise, hematology, serum chemistry, and blood gas analysis revealed trends comparable to human anthrax-related pathophysiology. The Dutch belted rabbit model of inhalational anthrax exhibited most of the physiologic, hematologic, and biochemical sequelae noted in human cases. Therefore, this rabbit model fulfills several of the criteria of a useful animal model for studying disease pathogenesis and evaluating therapeutics during inhalational anthrax.

Abbreviations: EdTx, edema toxin; LeTx, lethal toxin; MAP, mean arterial pressure; paCO2, arterial partial pressure of carbon dioxide; paO2, arterial partial pressure of oxygen; SaO2, arterial oxygen saturation; TCO2, total carbon dioxide

The etiologic agent of inhalational anthrax, Bacillus anthracis, continues to pique the interest of the scientific community because of its potential use as a biologic threat. This organism is classified as a category A priority pathogen, meaning an organism that poses a risk to national security because of its ease of dissemination and its ability to cause high mortality within a population. Anthrax bacteria produce 2 protein exotoxins: lethal toxin (LeTx) and edema toxin (EdTx).36 Individually and collectively, the toxins elicit a myriad of systemic complications; LeTx is composed of lethal factor and protective antigen, whereas EdTx is composed of edema factor and protective antigen. Lethal factor, a zinc metalloprotease, cleaves all mitogen-activated protein kinase kinases except isotype 5.6,15,30,38 In doing so, lethal factor interferes with numerous biologic processes that are regulated by signaling pathways involving these kinases.1,3,13 Edema factor is a calmodulin-dependent adenylyl cyclase that elevates the level of intracellular cAMP,6,15,30,38 an important second messenger also responsible for regulating many biological processes. Lastly, protective antigen is the receptor-binding moiety that, after heptamerization and vesicle acidification, facilitates entry of lethal factor and edema factor into the cytosol of target cells.8,30,38 When inhaled into the lungs, B. anthracis spores are engulfed within 6 h by alveolar macrophages23 and subsequently by dendritic cells,5,7 which migrate to mediastinal lymph nodes and release the germinated bacteria either by lysis or unknown mechanisms. After multiplication in the lymph nodes, the bacteria disseminate into the lymphatics, eventually navigating their way into the blood, where multiplication and spreading continues, resulting in bacteremia and, ultimately, death of the host.12,28,43

Previous animal studies of anthrax infection9,35,45-47 were conducted to analyze the physiologic effects after infection with B. anthracis. The goal of these studies often was to determine the factors contributing to mortality, and therefore, the key factors that should be addressed first and foremost during treatment. However, most of these studies were limited to the use of recombinant toxin proteins administered to the animal by either continuous infusion or parenteral injection.9,35,45-47 Although the treatment of animals with toxins often produces notable pathology and even death, it can cause physiologic reactions that are not congruent with those of bacterial infection. Recombinant LeTx and EdTx have profound, yet distinct, hemodynamic effects in rats when administered individually by either continuous infusion or single injection.9,47 Therefore, a challenge system involving the use of virulent anthrax spores that mimics natural infection is essential. Also crucial is an animal model that yields data that can be extrapolated to human cases. Nonhuman primates are thought to be the most desirable animal model for studying inhalational anthrax,19,20 but the high cost of the animals and inaccessibility to adequately equipped select agent laboratories are major limitations. A suitable alternative is the rabbit model, which has previously been used to examine the pathology associated with inhalational anthrax22 and to determine the efficacy of vaccines and antitoxic drugs against anthrax.31,48

In this study we investigated physiologic parameters of unrestrained, conscious Dutch belted rabbits infected with B. anthracis Ames spores by nasal instillation. In doing so, we hoped to demonstrate the usefulness of the Dutch belted rabbit as a model in studying inhalational anthrax infection. The physiologic parameters we continuously monitored included both hemodynamic and respiratory parameters as well as temperature. In addition, blood samples were collected at various time points and used for hematology, serum chemistry, and blood gas analysis. To our knowledge, this report is the first description of the overall systemic response to inhalation anthrax infection involving a rabbit model.

Materials and Methods

Animals.

Specific-pathogen-free female Dutch belted dwarf rabbits (n = 31; age, 7 to 8 wk; weight, 1.0 to 1.5 kg) were purchased from a commercial vendor (Myrtle's Rabbitry, Thompson Station, TN). The vendor's comprehensive health assessment, which includes serologic testing, indicated that the animals were free from Pasteurella multocida, Pasteurella pneumotropica, Bordetella bronchiseptica, Treponema cuniculi, Clostridium piliformis, cilia-associated respiratory bacillus, oral papillomavirus, arthropod ectoparasites, helminth endoparasites, and protozoans. On delivery, the animals were pair- or singly housed at the University of Texas Medical Branch Animal Resource Center in stainless steel, ventilated rabbit racks (Allentown, Allentown, NJ) and allowed to acclimate for 1 wk. Rabbits were fed approximately 170 g commercial chow (Rabbit Diet 5321, LabDiet, Richmond, IN) daily and given water ad libitum. The animal rooms were maintained on a 12:12-h light:dark cycle, with the temperature ranging from 19 to 22 °C and humidity between 30% and 70%. After surgical implantation and recovery (1 to 2 wk) and before infectious challenge, the animals were transferred to a select-agent–approved, restricted-access Animal Biosafety Level 3 laboratory and allowed to acclimate 24 h. The animals were singly caged in ventilated rabbit racks (Allentown), and environmental conditions were as previously described. All animal procedures were conducted under protocols approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee and Biological Safety Committee.

Preparation of B. anthracis spores.

Spores were prepared by inoculating B. anthracis Ames strain in Schaeffer sporulation medium [pH 7.0; 16 g Difco Nutrient Broth (Becton Dickinson, Franklin Lakes, NJ), 0.5 g MgSO4·7H2O, 2.0 g KCl, and 16.7 g MOPS per liter).41 Cultures were grown at 37 °C in 50-mL aliquots for 48 h, after which 100 mL sterile distilled water was added to dilute the medium and complete sporulation. After approximately 10 d of continuous shaking, sporulation of greater than 99% was confirmed by phase-contrast microscopy and by a modified Wirtz–Conklin spore stain.24 After centrifugation, the spore pellets were washed 4 times in sterile water and resuspended in sterile water. Subsequently, the spore suspension was layered onto a cushion of 58% Hypaque-76 (GE Healthcare, Piscataway, NJ) in a ratio of 1:2.5 (v/v). Mixing of the tubes was avoided, and they were centrifuged at 8270 × g for 45 min at 4 °C. The Hypaque supernatant was decanted, and the spore pellet was washed twice with sterile water and finally resuspended in sterile water. The stock spore suspension was diluted in water to the desired number of colony-forming units immediately before each animal challenge experiment. Serial plate counts were performed in triplicate to establish the concentration of viable spores in the spore stock vials (usually 1 × 109 to 1 × 1010 cfu/mL). B. anthracis cultures and spores were prepared and stored at −80 °C in a restricted-access Biosafety Level 2 laboratory registered with the Centers for Disease Control and Prevention and inspected by the Department of Defense and the United States Department of Agriculture.

Challenge procedure.

After collection for baseline data, rabbits were challenged with 100 LD50 of B. anthracis Ames spores (1 × 107 cfu) by using nasal instillation as described previously.40 For nasal instillation, the rabbits were anesthetized with ketamine (35 mg/kg; JA Webster Veterinary Supply, Denver, CO) and xylazine (5 mg/kg; JA Webster Veterinary Supply) by intramuscular injection. For nasal instillation of spores, anesthetized animals were suspended vertically by using the upper incisors, as described,40 with the bulk of the body weight of the rabbits resting on the base of the platform. The spore suspension was instilled slowly for 2 to 3 min into the anterior opening of each naris (50 μL each); then PBS (50 μL/naris) was used to wash any nonadherent spores from the nasal cavity into the lungs. All animal challenges were performed in an approved, restricted-access Animal Biosafety Level 3 laboratory under an approved Institutional Animal Care and Use Committee protocol.

Telemetry system.

The telemetry equipment (Data Sciences International, St Paul, MN) included an implantable transmitter (model TL11M3-D70-PCTP) that was customized to have 2 pressure leads for monitoring all pressure-related parameters and 2 biopotential leads for recording the electrocardiogram. Each system also included a wireless receiver (model RMC1) and a device that automatically relayed information regarding the receiver itself (Data Exchange Matrix). The software used for data analysis was Dataquest ART Platinum 4.1 (Data Sciences International).

Transmitter implantation.

Sterile transmitters were implanted in 3 rabbits by using aseptic technique. The rabbits were premedicated with glycopyrrolate (0.01 to 0.02 mg/kg SC; American Regent, Shirley, NJ), buprenorphine (0.01 to 0.05 mg/kg SC; Hospira, Lake Forest, IL), and flunixin meglamine (1.1 mg/kg IM; Schering-Plough Animal Health, Union, NJ). Anesthesia was induced by using ketamine (10 to 15 mg/kg IM; Fort Dodge Animal Health, Fort Dodge, IA) and diazepam (0.3 to 0.5 mg/kg IM; Hospira) and maintained by using 2% to 4% isoflurane (Webster Veterinary, Sterling, MA) delivered by facemask. The entire thoracic and abdominal regions and medial aspects of the hindlimbs were shaved and then scrubbed with povidone–iodine followed by isopropyl alcohol. The animals were placed on circulating warm-water pads to maintain normal body temperature.

A midline laparotomy was made at the middle 1/3 of the abdomen; additional incisions were made on the medial aspect of either thigh, and the femoral arteries were identified and isolated. One of the pressure transducers was exteriorized from the abdomen by using a 14-gauge needle as a guide to create a subcuticular channel. Three 3-0 Prolene (Ethicon, San Angelo, TX) stay sutures were placed underneath each isolated artery section (proximal occlusion, artery ligature and distal occlusion). A solution of 2% lidocaine (Phoenix Pharmaceutical, St Joseph, MO) was used to irrigate the femoral artery to dilate the artery and prevent vasospasms. Tension was applied to the proximal and distal sutures to occlude blood flow and to elevate the artery. The artery then was pierced with the bent tip of a 22-gauge needle cranial to the distal occlusion suture, and the catheter tip was inserted proximally into the vessel. The catheter tip was inserted beyond the iliac bifurcation and set in the abdominal aorta. The 3 stay sutures around the femoral artery then were used to tie down the catheter, holding it in the artery. The subcutaneous tissue on the medial aspect of the thigh was closed with 4-0 Vicryl (Ethicon), and the skin was closed with 3-0 Vicryl (Ethicon).

The second pressure lead was introduced into the pleural space by isolating the gastroesophageal junction and using a 22-gauge intravenous catheter to create a tunnel under the serosa of the esophagus to a point cranial to the diaphragm. The second pressure transducer was inserted into the tunnel and sutured in place with 4-0 Prolene (Ethicon). The electrocardiography leads were exteriorized from the abdomen just cranial to the laparotomy incision. A 2-cm skin incision was made on the cranial aspect of the right hemithorax. For placement of the negative lead, a trocar was used to create a subcutaneous tunnel from just cranial to the midline incision to the incision on the right hemithorax. The positive lead was placed on the caudal aspect of the thorax just cranial to the last rib. The subcutaneous tissue around both leads was closed with 4-0 Prolene, and the skin was closed with 3-0 Vicryl. For the midline laparotomy, the subcutaneous tissue and skin were closed with 3-0 Vicryl. Analgesia was accomplished by providing 0.5% bupivicaine (Hospira) in an infiltrative block at the incision site (midline laparotomy) once at closure; buprenorphine (0.01 to 0.05 mg/kg SC) was administered every 6 to 12 h as needed for the first 3 d. After surgery, the animals were monitored continually until they were ambulatory and a minimum of twice a day for the remainder of the recovery period. The need for analgesia during recovery was based on the animals' activity level, appearance, feeding behavior, and physiologic changes. The animals were allowed to recover 1 to 2 wk before infectious challenge.

Collection of telemetric data.

Parameters that were continuously monitored and recorded included mean arterial pressure (MAP), systolic pressure, diastolic pressure, pulse pressure, heart rate (HR) with electrocardiogram, respiratory rate, respiratory amplitude, inspiratory time, expiratory time, and temperature. Data collection began 24 h before anthrax challenge (baseline levels) and continued until death. Death was used as an endpoint in light of the variability in time to death, absence of any reliable markers of impending death, and absence of any visible signs of illness. The rabbits did not become moribund prior to death, which occurred suddenly and without warning. Data collection was stopped during anesthesia and intranasal challenge but began again immediately thereafter. The data acquisition system was programmed to record data every 60 s. These data were used to compute either 2- or 4-h moving averages. The computed numerical data of the moving averages were exported to an Excel spreadsheet (Microsoft, Redmond, WA) for graph preparation.

Hematology and serum chemistry.

Four anthrax-challenged rabbits were bled before (0 h; that is, baseline) and after challenge (12, 24, and 48 h) from the central artery or marginal vein of the ear. The central artery was the primary route for obtaining samples (approximately 1 mL each), but the marginal vein was used as an alternative if blood could not be obtained from the central artery, which happened only rarely. At 60 h after challenge, the animals were bled by cardiocentesis, which was a terminal procedure. In the case of terminal blood samples, the animals first were anesthetized with ketamine (35 mg/kg) and xylazine (5 mg/kg) by intramuscular injection, bled by cardiocentesis, and euthanized by injection of pentobarbital sodium (1 mL) directly into the heart, with cutting of the animals' diaphragms after cessation of respiration to assure death. Blood samples intended for serum analysis were refrigerated overnight in serum-separator tubes (Microtainer Amber Tubes with Serum Separator, Becton Dickinson). Serum concentrations of aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, alkaline phosphatase, albumin, calcium, creatine kinase, potassium, and glucose were measured by using an automated analyzer (Prochem V, Drew Scientific, Oxford, CT). Blood samples intended for hematologic analyses, stored in anticoagulant tubes (Microtainer Brand Tube with K2EDTA, Becton Dickinson), were performed within 2 h of blood collection by using an automated hematology system (Hemavet 950, Drew Scientific). The hematologic analyses included measurements for WBC concentration, neutrophil concentration, lymphocyte concentration, RBC concentration, hematocrit, platelet concentration, monocyte concentration, basophil concentration, eosinophil concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, and red cell distribution width.

Blood analysis.

Blood samples (0.3 to 0.5 mL) from 24 rabbits were collected from the central artery of the ears before (0 h; that is, baseline level) and after challenge. Arterial blood was also collected every 12 h beginning at 60 h after challenge and continued until the death of the animal. These times were selected to avoid collapsed arteries due to multiple bleeds and to acquire measurements late in infection, when biochemical changes were imminent. Immediately after collection, whole blood was analyzed by using a portable clinical analyzer (iSTAT, Heska Corporation, Fort Collins, CO), which measured blood gases (including HCO3, base excess, and pH; n = 5) and lactate levels (n = 4) as well as activated clotting time (n = 6) and cardiac troponin I (n = 9). Blood collection for cardiac troponin I began at 48 h after infectious challenge, and some samples collected for measurement of this cardiac marker were extracted from the marginal vein of the ear.

Statistical analysis.

Each variable was evaluated separately for significant temporal changes from baseline by using 2-way ANOVA. The animal × time interaction mean square was used for all significance tests. The type I error rate for each variable's analysis was set at 0.05 or less. Data were entered and maintained in Excel (Microsoft) and imported into the statistical package NCSS (NCSS, Kaysville, UT) for statistical analyses. The times of measurement for each variable were as described elsewhere. When ANOVA showed significant temporal changes, the baseline average was compared with the average at each subsequent time period by using Dunnett posthoc tests. Means that had significant changes (prespecified increases or decreases) from baseline, based on the results of the Dunnett tests, are so indicated in the figure legends, along with their standard deviations and sample sizes. The use of these statistical analysis procedures adjusts the significance levels to account for multiple testing.

Results

Physiologic responses to infection.

Figure 1 A through G illustrates the physiologic responses of 3 rabbits to inhalational anthrax. The sharp dip that begins at 0 h and continues until 8 h in some graphs denotes the time during which the animals were anesthetized, challenged, and allowed to recover from anesthesia. Pressure and temperature data are presented as 2-h moving averages, and respiratory data are given as 4-h moving averages. All rabbits experienced mild tachycardia (heart rate greater than 300 beats per minute42) during infection but at different times. Before death, 2 of the 3 animals (rabbits 2 and 3) also exhibited a slightly depressed heart rate (at 56 h after challenge) subsequent to their tachycardic responses (Figure 1 A). Even though the heart rate for rabbit 2 exceeded the normal physiological range (200 to 300 beats per minute42) at baseline and immediately after challenge, a notable increase above baseline still was present at 45 h after challenge. Electrocardiography (data not shown) confirmed the presence of tachycardia during infection and bradycardia (heart rate less than 200 beats per minute42) during the agonal phase. All the animals became consistently hypotensive (MAP less than 83 mm Hg42) during late infection and before death, although 2 animals (rabbits 2 and 3) displayed a slight increase in MAP (at 42 h after challenge) before this decline (Figure 1 B). The MAP of rabbit 3 was below the lower limit for normal rabbits for approximately 24 h after challenge, but it rebounded and appeared to stabilize for a while before the agonal phase. The trend with pulse pressure was a steady decline, which began approximately 1 to 2 d before the death of the animals (Figure 1 C). The trends in the systolic and diastolic pressures (not shown) were similar to that of MAP, but systolic pressure fell slightly faster than did diastolic pressure and therefore the waning pulse pressure.

Figure 1.
Hemodynamic and respiratory parameters and temperature. (A) Heart rate (in beats per minute, bpm), (B) mean arterial pressure (MAP), (C) pulse pressure, (D) respiratory rate (in respiratory cycles per minute, rcpm), (E) inspiratory time, (F) peak amplitude, ...

With regard to respiration, all 3 rabbits were consistently tachypneic (respiration rate greater than 60 breaths per minute42) hours before death (Figure 1 D). The respiration rates for rabbits 1 and 2 were only slightly above the normal physiologic range (30 to 60 breaths per minute42) at baseline and at times during midinfection, but the most dramatic changes did not occur until late in infection. Two of the 3 animals (rabbits 2 and 3) had notable temporary declines in inspiratory time at approximately 44 h after challenge, with the remaining animal showing a decline at 6 h after challenge and a less notable decline at approximately 60 h (Figure 1 E). Unlike the inspiratory time, expiratory time showed a transient increase late in infection, primarily in only 1 animal (data not shown). The respiratory amplitude, which is indicative of the strength of inspiration, rose in intensity (became more negative) for several hours at approximately 42 h after challenge in 2 of the 3 animals (rabbits 1 and 2; Figure 1 F). All 3 rabbits exhibited a transient febrile response (above 40 °C42), which persisted for less than 24 h (Figure 1 G).

Hematology.

As can be expected at the onset of a bacterial infection, the 4 rabbits evaluated exhibited a statistically significant (P < 0.05) increase in WBC concentration between 0 and 24 h and between 0 and 48 h after anthrax challenge (Figure 2 A). The increase in WBC for 3 of the 4 rabbits was greater than 80%. By 60 h after challenge, the WBC concentration returned to baseline level. The fluctuations in WBC concentrations can largely be attributed to the changes in the rabbits' neutrophil concentrations. From 0 to 24 and 48 h, the animals demonstrated significant increases in neutrophil counts (Figure 2 B), but like the WBC count, the neutrophil concentration fell back to baseline level at 60 h postchallenge. Based on the similarity in trends, changes in lymphocyte concentration (data not shown) may have contributed to the overall fluctuation in the WBC level even though the variations in lymphocyte concentration did not reach statistical significance themselves. Otherwise, measured monocyte, basophil, and eosinophil concentration values were relatively stable for the duration of the experiment (data not shown).

Figure 2.
Hematology. (A) WBC concentration, (B) neutrophil concentration, (C) RBC concentration, and (D) hematocrit of rabbits (n = 4) before and during anthrax infection. Rabbits were challenged with 100 LD50 Ames spores. Blood was collected from the central ...

The rabbits' intranasal instillation with anthrax also affected their erythrocytes. The 4 rabbits experienced a significant (P < 0.05) decrease in RBC concentrations by 60 h after challenge, most of which exhibited a decline of approximately 30% (Figure 2 C). With regard to hematocrit, the rabbits demonstrated a statistically significant (P < 0.05) decline from 0 to 60 h postchallenge (Figure 2 D). The rabbits' mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, and red cell distribution width did not change significantly after infectious challenge (data not shown). In addition, B. anthracis lacked any significant effect on platelet concentration and mean platelet volume (data not shown).

Serum chemistry.

The alanine aminotransferase level showed a significant (P < 0.05) increase that began 12 h after challenge and persisted thereafter (Figure 3 A). Differences in serum aspartate aminotransferase levels did not reach significance (data not shown). Glucose concentrations rose during infection, but not until 60 h after challenge (Figure 3 B), when it had increased by more than 60%. Furthermore, within the first 12 h after challenge and throughout infection, the rabbits exhibited dramatic elevations in creatine kinase concentration, with increases of greater than 200% at some time points (Figure 3 C). Data collected regarding blood urea nitrogen, alkaline phosphatase, albumin, calcium, and potassium did not demonstrate any significant trends (data not shown).

Figure 3.
Serum chemistry. (A) Alanine aminotransferase (ALT), (B) glucose, and (C) creatine kinase in rabbits (n = 4) before and during anthrax infection. Rabbits were challenged with 100 LD50 Ames spores. Blood was collected from the central artery or marginal ...

Blood analysis.

Beginning at 60 h after challenge, the animals' arterial partial pressure of oxygen (paO2) declined significantly (P < 0.05; Table 1). Arterial oxygen saturation (SaO2) also declined, but the decline was significant (P < 0.05) only at the last collection time, which was within 12 h of death of the animal (Table 1). The change in arterial partial pressure of carbon dioxide (paCO2) at the last collection time did not reach significance (Table 1). At 60 h after challenge and later, total CO2 (TCO2), HCO3, base excess, and blood pH were significantly (P < 0.05) decreased compared with baseline levels (Table 1). For these particular parameters and paO2, the mean values at 60 h and the last collection point were statistically indistinguishable. Furthermore, comparing levels before infection with those within 12 h before death revealed significant (P < 0.05) increases in blood lactate, activated clotting time, and cardiac troponin I (Table 2). Only data for the latest time point available are presented for these 3 parameters, due to either minimal changes midinfection or differences in time to death between animals.

Table 1.
Arterial blood gas analysis of 5 rabbits before and after anthrax challenge
Table 2.
Blood analysis of rabbits before and after anthrax challenge

Discussion

To our knowledge, the present study is the first to give a comprehensive account of the physiologic responses to inhalational anthrax infection in a rabbit model. With the use of continuous telemetric monitoring, we were able to witness hemodynamic and respiratory abnormalities that could easily be overlooked due to their transience. This monitoring, along with hematology, serum chemistry, and blood gas analysis, allowed us to describe thoroughly the overall response to inhalational anthrax in the rabbit model. The rabbit response parallels many of the features of human cases of anthrax. Although knowing the effects caused by toxins alone is quite valuable to characterizing anthrax infection, being aware of the overall effects due to inhalation of anthrax spores by using a reliable and suitable animal model is equally important.

Our results suggest that anthrax infection has profound hemodynamic effects in the rabbit model, and these effects may be key to the bacteria's ability to cause lethality. All the rabbits developed tachycardia during mid- to late infection and refractory bradycardia and asystole at the end. Purified anthrax toxins previously were described to have cardiac effects, but reports conflicted. According to some groups, EdTx increased the heart rate of rats during continuous infusion,9,47 but others found it decreased the heart rate of mice.17 Furthermore, some reported that LeTx brought about a dramatic bradycardic response in rats, but according to others, it had little to no effect on heart rate.9,47 We speculate that differences in cardiac response between our model system and those of previous studies using purified toxins are due to the presence of whole bacteria. Animals challenged with whole B. anthracis must contend not only with the toxins produced by the bacteria but also with the bacteria's structural components, which include a capsule, S-layer, and peptidoglycan layer.36 All of these components induce immune responses2,21,27 that could have physiologic effects on the host. The increases in cardiac troponin I, creatine kinase, and glucose that we witnessed further imply that anthrax infection caused cardiac damage, a feature previously reported to occur in mice challenged with recombinant EdTx.17 According to histopathologic analysis, the cardiac tissue of the mice in the cited study had extensive areas of cardiomyocytic necrosis at 36 and 48 h after challenge, but some damage could be due to tissue ischemia nevertheless. Also worth noting from our observations is the escalating heart rate concomitant with the diminishing pulse pressure, which is suggestive of decreased stroke volume. This hypothesis is supported in part by the findings that LeTx and EdTx respectively reduced left ventricular systolic function and preload.46 Nonetheless, given the close link between heart rate and blood pressure, the changes in heart rate that we noted may be secondary to changes in blood pressure and not due to the toxins specifically. Moreover, the increases may indicate the incidence of systemic inflammatory response syndrome, which is the precursor to septic shock and which occurs in other bacterial infections.33,39

Another consistent finding in the present study is the hypotensive state that began midinfection. When presented at similar lethal doses, LeTx and EdTx continuously infused into rats for 24 h also evoked a decrease in mean blood pressure, with EdTx showing the greater effect.9,10 The toxic effects on blood pressure can be explained by both the ability of LeTx to induce endothelial barrier dysfunction leading to increased vascular permeability and decreased systemic vascular resistance45 and by the ability of EdTx to dramatically increase cAMP levels, which can reduce systemic vascular resistance.29 Hemorrhaging is common with anthrax infection and therefore is another plausible explanation for the observed hypotension.11,19,22,40,49 The decreases in RBC count and hematocrit (with a normal distribution in red cell width) that we observed suggest that the animals were hemorrhaging. Importantly, along with hemorrhaging often comes metabolic acidosis, specifically type A lactic acidosis, a disorder that arises when the body's tissues are poorly perfused. The reductions in TCO2, HCO3, base excess, and blood pH along with the increase in lactate strongly suggest that the animals underwent this type of acidosis before death.

Due to the consistent trend in MAP late in infection, this physiologic parameter potentially can serve as an indicator of imminent death in future studies using the rabbit model of inhalational anthrax. Currently, there are no illness models in the case of inhalational anthrax, and there have been no precise markers of impending death. The Dutch belted rabbit shows no obvious physical signs of illness prior to death. The animals exhibit normal physical appearance, eating habits, and behavior throughout infection until death. Consequently, predicting death by mere observation is highly improbable. However, by continuously monitoring the animals by using telemetry, we find that it is possible to construe a fall in MAP as an indicator of impending death. Comparing MAP at baseline to that immediately prior to the agonal phase revealed a 22% to 25% decrease that persisted for 18 to 24 h. After this decline, there was no rebound in the MAP. Therefore, in future experiments involving B. anthracis-infected animals with implanted transponders, MAP might be used as an endpoint predictive of impending death and indicative of euthanasia. However, this association must be confirmed by using a larger number of animals.

The data suggest that spore-induced anthrax infection brought about prominent respiratory changes consistent with respiratory distress in this model. Hours before death, all the animals became tachypneic, a situation common to systemic inflammatory response syndrome. With 2 animals, the inspiratory time fell below baseline levels suggesting that at these time points, the animals were either inspiring less or gasping rapidly. We suspect that the decline at 6 h after challenge in rabbit 1, which began immediately after challenge and too soon for significant bacterial proliferation, was most likely due to an unusual response to anesthesia rather than to the infection itself. The increased inspiratory intensity as shown by the respiratory amplitude of the remaining 2 rabbits supports the idea of gasping. This finding, together with the deficiencies in paO2 and SaO2, suggest the animals undergo severe respiratory distress late in infection. In addition, the animals that showed greater hypoxemia (defined as paO2 less than 85 mm Hg42) and larger increases in paCO2 displayed more dramatic reductions in blood pH. This pattern suggests that, in addition to having metabolic acidosis, these animals were undergoing respiratory acidosis, which is distinguished by the increase in paCO2. Hypoxemia, pleural effusion, and pulmonary edema are all consistently associated with inhalational anthrax infection,18,26 and together they indicate the presence of acute lung injury.

The transient febrile response in our rabbits implies a systemic cytokine response during infection, despite previous reports showing that LeTx and EdTx inhibit the production of cytokines including IL1 and TNFα,16,25,44 both of which have a role in the induction of fever.32 Perhaps the fever is lessened only after the bacteria have produced a sufficient amount of toxin capable of inhibiting systemic cytokine production. A previous study37 demonstrated that the production of some cytokines in mice similarly is transient during infection. This association would explain in part a transitory febrile response in the midst of ongoing anthrax infection. Then again, the fact that rabbits challenged with a larger dose of Ames spores (300 LD50) had a prolonged fever (data not shown) discounts this assumption and suggests that transient fever is simply a phenomenon associated with sepsis.

Inhalation anthrax infection was associated with hematologic abnormalities in these rabbits. The WBC count increased midinfection, as is common for bacterial infections, but the levels fell back to or below baseline levels late in infection. This decline is attributed in large part to the waning neutrophil response that we observed from 48 to 60 h after challenge. In the mouse model, anthrax toxins previously were shown to work together to enhance neutrophil apoptosis.14 Our results regarding activated clotting time suggest that the animals also had a deficiency in blood clotting. The potential damage to the liver (as suggested by the increased alanine aminotransferase level and slight rise in aspartate aminotransferase concentration), the organ responsible for the production of various clotting factors, and the potential reduction in platelet count support this conclusion.

Many of the aforementioned physiologic responses in the present study are analogous to those reported in human cases of inhalational anthrax infection.4,26,34 Patients who became infected experienced tachycardia, regular and irregular, followed by bradycardia and eventually asystole. Some of these patients also developed atrial fibrillation with variable ventricular disturbances. Similar to our findings, patients experienced hypotension during late infection but this was not present on admission in every case. The pathology report regarding the Sverdlovsk epidemic of 1979 described the occurrence of low- to high-pressure hemorrhage in all cases,22 as was suggested by the hematology results we obtained. Occasional persons had metabolic acidosis,4,26 which was most likely due to poor tissue perfusion from hemorrhaging and hypotension. The majority of reported cases showed increases in respiratory rate with signs of respiratory distress and hypoxemia,4,26,34 all of which we witnessed in our rabbit model. Lastly, some patients developed coagulopathies,4,34 as indicated by prolonged prothombin time and partial thromboplastin time (we used activated clotting time), and all patients became febrile and had elevated hepatic transaminases.4,26,34 All of these events occurred in the present study.

In summary, we found that rabbits undergoing inhalation anthrax infection initiated by instillation of virulent B. anthracis spores have physiologic responses comparable to responses in humans. Although additional pathophysiology associated with anthrax infection remains to be compared between humans and rabbits, the similarities presented here highlight the usefulness of the rabbit as a suitable model for studying inhalation anthrax infection.

Acknowledgments

This work was supported by the US Army (DAMD170210699) and the National Institute of Health (NO1-AI-30065). Bacillus anthracis Ames was generously provided by C Richard Lyons (University of New Mexico Health Science Center, Albuquerque, NM) and duly registered with the Centers for Disease Control.

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