Recombinant protective antigen anthrax vaccine improves survival when administered as a postexposure prophylaxis countermeasure with antibiotic in the New Zealand white rabbit model of inhalation anthrax.

Inhalation anthrax is a potentially lethal form of disease resulting from exposure to aerosolized Bacillus anthracis spores. Over the last decade, incidents spanning from the deliberate mailing of B. anthracis spores to incidental exposures in users of illegal drugs have highlighted the importance of developing new medical countermeasures to protect people who have been exposed to "anthrax spores" and are at risk of developing disease. The New Zealand White rabbit (NZWR) is a well-characterized model that has a pathogenesis and clinical presentation similar to those seen in humans. This article reports how the NZWR model was adapted to evaluate postexposure prophylaxis using a recombinant protective antigen (rPA) vaccine in combination with an oral antibiotic, levofloxacin. NZWRs were exposed to multiples of the 50% lethal dose (LD(50)) of B. anthracis spores and then vaccinated immediately (day 0) and again on day 7 postexposure. Levofloxacin was administered daily beginning at 6 to 12 h postexposure for 7 treatments. Rabbits were evaluated for clinical signs of disease, fever, bacteremia, immune response, and survival. A robust immune response (IgG anti-rPA and toxin-neutralizing antibodies) was observed in all vaccinated groups on days 10 to 12. Levofloxacin plus either 30 or 100 µg rPA vaccine resulted in a 100% survival rate (18 of 18 per group), and a vaccine dose as low as 10 µg rPA resulted in an 89% survival rate (16 of 18) when used in combination with levofloxacin. In NZWRs that received antibiotic alone, the survival rate was 56% (10 of 18). There was no adverse effect on the development of a specific IgG response to rPA in unchallenged NZWRs that received the combination treatment of vaccine plus antibiotic. This study demonstrated that an accelerated two-dose regimen of rPA vaccine coadministered on days 0 and 7 with 7 days of levofloxacin therapy results in a significantly greater survival rate than with antibiotic treatment alone. Combination of vaccine administration and antibiotic treatment may be an effective strategy for treating a population exposed to aerosolized B. anthracis spores.

A short course of antibiotic treatment is effective in preventing death from experimental inhalational anthrax after discontinuing antibiotics.

BACKGROUND: Postexposure prophylaxis of inhalational anthrax requires prolonged antibiotic therapy or antibiotics and vaccination. The duration of treatment for established anthrax is controversial, because retained spores may germinate and cause disease after antibiotics are discontinued. Using rhesus macaques, we determined whether a short course of antibiotic treatment, as opposed to prophylaxis, could effectively treat inhalational anthrax and prevent disease caused by the germination of spores after discontinuation of antibiotics. METHODS: Two groups of 10 rhesus macaques were exposed to an aerosol dose of Bacillus anthracis spores. Animals in group 1 received ciprofloxacin prophylaxis beginning 1-2 h after exposure. Those in group 2 began receiving ciprofloxacin after becoming bacteremic, and treatment was continued for 10 days. When each group 2 animal completed 10 days of therapy, the prophylactic antibiotic was discontinued in the paired group 1 animal. RESULTS: In group 1 (prophylaxis), no deaths occurred during antibiotic treatment, but only 2 (20%) of 10 animals survived after antibiotics were discontinued. In contrast, in group 2 (treatment), 3 deaths occurred during antibiotic treatment, but all 7 animals (100%) alive after 10 days of therapy survived when antibiotics were discontinued. CONCLUSIONS: In the treatment of inhalational anthrax, the prolonged course of antibiotics required to achieve prophylaxis may not be necessary to prevent anthrax that results from the germination of retained spores after the discontinuation of antibiotics.

Whole-body plethysmography in African green monkeys (Chlorocebus aethiops) with and without jackets.

Indwelling central venous catheters are often used to facilitate frequent phlebotomy while minimizing stress and anesthetic effects on animals. However, nonhuman primates with central venous catheters must wear protective jackets. Jackets routinely are removed for aerosol exposure to agents and respiratory measurements by whole-body plethysmography (WBP) because of the potentially confounding effects of jackets on these procedures. However, removing the jacket may dislodge the catheter, making it unusable. Using each animal as its own control, we tested 12 African green monkeys to determine whether minute volume, tidal volume, respiratory rate, or accumulated volume measurements by WBP differed depending on whether the animal wore a protective jacket or not. We found no statistical differences in any measured respiratory parameter and concluded that the jackets could be left in place on the animal while undergoing plethysmography without compromising the calculations for determining the inhaled dose of aerosolized agent. In addition, this study revealed no obvious contraindications to leaving the jacket in place in other nonhuman primate species, provided that the jacket fits appropriately and that plethysmography is performed correctly.

Identification of a surrogate marker for infection in the African green monkey model of inhalation anthrax.

In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.

Nosocomial infection of Serratia marcescens may induce a protective effect in monkeys exposed to Bacillus anthracis.

This study was originally designed to collect data on the natural history of inhalational anthrax in a new nonhuman primate model. An uncontrollable event created a new experimental condition which allowed us to retrospectively evaluate the power of the innate immune system to protect from an aerosol exposure of B. anthracis. Five African green monkeys (AGMs) had intravenous catheters implanted. One catheter was accidentally pulled out, leaving four AGMs with catheters and one without. All were exposed, to multiple lethal doses of B. anthracis Ames strain. Blood was collected twice daily to evaluate bacteremia. The AGM with no catheter had blood drawn from a femoral vein and became bacteremic on Day 9; succumbed to inhalational anthrax on Day 10. The other four AGMs had S. marcescens contamination in the catheter; indicated by pure colonies grown from the blood. None of these AGMs showed clinical signs of illness, had B. anthracis or a detectable level of protective antigen in the bloodstream. It appears that the presence of S. marcescens may have induced a "Coley's toxin" effect in this experiment. The innate immune response may have protected the AGMs from a lethal inhalational dose of B. anthracis spores.

Use of a low-concentration heparin solution to extend the life of central venous catheters in African green monkeys (Chlorocebus aethiops).

Normal hematologic values for African green monkeys have been reported, but these results are confounded by the effect of chemical restraint (for example, ketamine), physical restraint, and capture stress. The dual-lumen central venous catheter, jacket, and tether combination we describe here allows intravenous fluid administration and repeated blood sampling without the use of anesthesia or inducing capture-related stress. The use of a low-concentration heparin solution for catheter maintenance significantly increased the mean patency time, compared with a saline-only catheter flush solution. Adding a low-concentration heparin solution creates a suitable system for serial blood collection in the African green monkey for as long as 25 d.

Short-course postexposure antibiotic prophylaxis combined with vaccination protects against experimental inhalational anthrax.

Prevention of inhalational anthrax after Bacillus anthracis spore exposure requires a prolonged course of antibiotic prophylaxis. In response to the 2001 anthrax attack in the United States, approximately 10,000 people were offered 60 days of antibiotic prophylaxis to prevent inhalational anthrax, but adherence to this regimen was poor. We sought to determine whether a short course of antibiotic prophylaxis after exposure could protect non-human primates from a high-dose spore challenge if vaccination was combined with antibiotics. Two groups of 10 rhesus macaques were exposed to approximately 1,600 LD50 of spores by aerosol. Both groups were given ciprofloxacin by orogastric tube twice daily for 14 days, beginning 1-2 h after exposure. One group also received three doses of the licensed human anthrax vaccine (anthrax vaccine adsorbed) after exposure. In the ciprofloxacin-only group, four of nine monkeys (44%) survived the challenge. In contrast, all 10 monkeys that received 14 days of antibiotic plus anthrax vaccine adsorbed survived (P = 0.011). Thus postexposure vaccination enhanced the protection afforded by 14 days of antibiotic prophylaxis alone and completely protected animals against inhalational anthrax. These data provide evidence that postexposure vaccination can shorten the duration of antibiotic prophylaxis required to protect against inhalational anthrax and may impact public health management of a bioterrorism event.

Marburg and Ebola viruses as aerosol threats.

Ebola and Marburg viruses are the sole members of the genus Filovirus in the family Filoviridae. There has been considerable media attention and fear generated by outbreaks of filoviruses because they can cause a severe viral hemorrhagic fever (VHF) syndrome that has a rapid onset and high mortality. Although they are not naturally transmitted by aerosol, they are highly infectious as respirable particles under laboratory conditions. For these and other reasons, filoviruses are classified as category A biological weapons. However, there is very little data from animal studies with aerosolized filoviruses. Animal models of filovirus exposure are not well characterized, and there are discrepancies between these models and what has been observed in human outbreaks. Building on published results from aerosol studies, as well as a review of the history, epidemiology, and disease course of naturally occurring outbreaks, we offer an aerobiologist's perspective on the threat posed by aerosolized filoviruses.

Drinking water exposure to cadmium, an environmental contaminant, results in the exacerbation of autoimmune disease in the murine model.

Cadmium is a pervasive environmental contaminant. The primary route of exposure to the general population occurs via contaminated drinking water or food supplies. Our hypothesis was that cadmium could be a trigger for inducing autoimmune disease (AD) in genetically predisposed populations. Therefore, New Zealand Black/White F1 (NZBW) mice were exposed to cadmium via drinking water. Mice were exposed to: 0, 3, 30, 3000 or 10000 parts per billion (ppb) of cadmium in tap water for 2, 4, 28, or 31 weeks. After 4 weeks of exposure, in the group of mice exposed to 10000 ppb cadmium, there was an increased incidence of antinuclear antibodies (ANA). There was also deposition of immune complexes in all groups after 4 weeks of exposure. After 31 weeks, there were increases in IgG2a in mice exposed to low doses of cadmium. In an attempt to establish the progression from an autoimmune reaction to the development of AD, the biological marker for AD, proteinuria, was assessed. Onset of proteinuria was exacerbated by 11 weeks in mice exposed to cadmium. This data suggests that short-term exposure may result in a type of autoimmune reaction since the mice are beginning to produce ANA after only 4 weeks of exposure and there is immune-complex deposition in the kidney. Long-term exposure to cadmium appears to result in the exacerbation of AD as indicated by the development of proteinuria and continued presence of immune complexes in the kidney. The mechanism may involve the increased production of IgG2a, which is capable of forming immune complexes and causing autoimmune glomerulonephritis.