Application of Bacillus anthracis PCR to simulated clinical samples

We evaluated PCR for the detection of Bacillus anthracis DNA from simulated clinical specimens relevant for the microbiological diagnosis of anthrax or exposure to B. anthracis spores. In simulated blood specimens, the lowest limit of detection was 400 CFU per mL of blood, which may be sufficient for samples from patients with septic anthrax. Screening nasal swabs by PCR may not be sensitive enough to rule out dangerous exposure to anthrax spores, as a minimum of 2000 spores per sample was required for detectable amplification. As spores survived some standard DNA purification methods, special attention should be paid to laboratory safety when preparing samples possibly containing live spores.     

Morphologic, immunologic, and molecular methods to detect bacillus anthracis in formalin-fixed tissues.

Due to the importance of Bacillus anthracis as a cause of naturally occurring infection among humans and as an agent of bioterrorism, there is a vital need for rapid and specific assays, including immunohistochemistry (IHC) and polymerase chain reaction (PCR) assays, to detect the bacterium in formalin-fixed tissues. Colorimetric IHC assays were developed using a multistep indirect immunoalkaline phosphatase method with anti-B. anthracis cell wall (EAII-6G6-2-3) and anti-B. anthracis capsule (FDF-1B9) mAbs to detect B. anthracis antigens in formalin-fixed, paraffin-embedded bacterial cultures and tissues. B. anthracis antigens were localized, using both antibodies, in samples from B. anthracis-infected animals and humans. The colorimetric IHC assay with both antibodies was expedient in diagnosing the presence of B. anthracis in formalin-fixed, paraffin-embedded tissue from bioterrorism-associated cases of inhalational and cutaneous anthrax and from a case of naturally occurring cutaneous anthrax. Using the same antibodies, confocal microscopy demonstrated the structure of replicating B. anthracis in tissues. B. anthracis-specific primers were successfully used with PCR to amplify and detect B. anthracis sequences derived from formalin-fixed tissues of anthrax cases. In this study, morphologic, immunologic, and molecular assays were used to study and diagnose 22 veterinary and human anthrax cases.     

Antibacterial role for natural killer cells in host defense to Bacillus anthracis

Natural killer (NK) cells have innate antibacterial activity that could be targeted for clinical interventions for infectious disease caused by naturally occurring or weaponized bacterial pathogens. To determine a potential role for NK cells in immunity to Bacillus anthracis, we utilized primary human and murine NK cells, in vitro assays, and in vivo NK cell depletion in a murine model of inhalational anthrax. Our results demonstrate potent antibacterial activity by human NK cells against B. anthracis bacilli within infected autologous monocytes. Surprisingly, NK cells also mediate moderate antibacterial effects on extracellular vegetative bacilli but do not have activity against extracellular or intracellular spores. The immunosuppressive anthrax lethal toxin impairs NK gamma interferon (IFN-γ) expression, but neither lethal nor edema toxin significantly alters the viability or cytotoxic effector function of NK cells. Compared to human NK cells, murine NK cells have a similar, though less potent, activity against intracellular and extracellular B. anthracis. The in vivo depletion of murine NK cells does not alter animal survival following intranasal infection with B. anthracis spores in our studies but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrate that NK cells participate in the innate immune response against B. anthracis and suggest that immune modulation to augment NK cell function in early stages of anthrax should be further explored in animal models as a clinical intervention strategy.     

Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays

Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.     

In vivo demonstration and quantification of intracellular Bacillus anthracis in lung epithelial cells

Inhalational anthrax is initiated by the entry of Bacillus anthracis spores into the lung. A critical early event in the establishment of an infection is the dissemination of spores from the lung. Using in vitro cell culture assays, we previously demonstrated that B. anthracis spores are capable of entering into epithelial cells of the lung and crossing a barrier of lung epithelial cells without apparent disruption of the barrier integrity, suggesting a novel portal for spores to disseminate from the lung. However, in vivo evidence for spore uptake by epithelial cells has been lacking. Here, using a mouse model, we present evidence that B. anthracis spores are taken up by lung epithelial cells in vivo soon after spores are delivered into the lung. Immunofluorescence staining of thin sections of lungs from spore-challenged BALB/c mice revealed that spores were associated with the epithelial surfaces in the airway and the alveoli at 2 and 4 h postinoculation. Confocal analysis further indicated that some of the associated spores were surrounded by F-actin, demonstrating intracellular localization. These observations were further confirmed and substantiated by a quantitative method that first isolated lung cells from spore-challenged mice and then stained these cells with antibodies specific for epithelial cells and spores. The results showed that substantial amounts of spores were taken up by lung epithelial cells in vivo. These data, combined with those in our previous reports, provided powerful evidence that the lung epithelia were directly targeted by B. anthracis spores at early stages of infection.     

Murine aerosol challenge model of anthrax

The availability of relevant and useful animal models is critical for progress in the development of effective vaccines and therapeutics. The infection of rabbits and non-human primates with fully virulent Bacillus anthracis spores provides two excellent models of anthrax disease. However, the high cost of procuring and housing these animals and the specialized facilities required to deliver fully virulent spores limit their practical use in early stages of product development. Conversely, the small size and low cost associated with using mice makes this animal model more practical for conducting experiments in which large numbers of animals are required. In addition, the availability of knockout strains and well-characterized immunological reagents makes it possible to perform studies in mice that cannot be performed easily in other species. Although we, along with others, have used the mouse aerosol challenge model to examine the outcome of B. anthracis infection, a detailed characterization of the disease is lacking. The current study utilizes a murine aerosol challenge model to investigate disease progression, innate cytokine responses, and histological changes during the course of anthrax after challenge with aerosolized spores. Our results show that anthrax disease progression in a complement-deficient mouse after challenge with aerosolized Sterne spores is similar to that described for other species, including rabbits and non-human primates, challenged with fully virulent B. anthracis. Thus, the murine aerosol challenge model is both useful and relevant and provides a means to further investigate the host response and mechanisms of B. anthracis pathogenesis.     

Progress and novel strategies in vaccine development and treatment of anthrax

The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.     

Protective antigen as a correlative marker for anthrax in animal models

The most aggressive form of anthrax results from inhalation of airborne spores of Bacillus anthracis and usually progresses unnoticed in the early stages because of unspecific symptoms. The only reliable marker of anthrax is development of bacteremia, which increases with disease progress. Rapid diagnosis of anthrax is imperative for efficient treatment and cure. Herein we demonstrate that the presence and level of a bacterial antigen, the protective antigen (PA), a component of B. anthracis toxins, in host sera can serve as a reliable marker of infection. This was tested in two animal models of inhalation anthrax, rabbits and guinea pigs infected by intranasal instillation of Vollum spores. In both models, we demonstrated qualitative and quantitative correlations between levels of bacteremia and PA concentrations in the sera of sick animals. The average time to death in infected animals was about 16 h after the appearance of bacteremia, leaving a small therapeutic window. As the time required for immunodetection of PA can be very short, the use of this marker will be beneficial for faster diagnosis and treatment of inhalation anthrax.     

Experimental respiratory anthrax infection in the common marmoset (Callithrix jacchus)

Inhalational anthrax is a rare but potentially fatal infection in man. The common marmoset (Callithrix jacchus) was evaluated as a small non-human primate (NHP) model of inhalational anthrax infection, as an alternative to larger NHP species. The marmoset was found to be susceptible to inhalational exposure to Bacillus anthracis Ames strain. The pathophysiology of infection following inhalational exposure was similar to that previously reported in the rhesus and cynomolgus macaque and humans. The calculated LD(50) for B. anthracis Ames strain in the marmoset was 1.47 x 10(3) colony-forming units, compared with a published LD(50) of 5.5 x 10(4) spores in the rhesus macaque and 4.13 x 10(3) spores in the cynomolgus macaque. This suggests that the common marmoset is an appropriate alternative NHP and will be used for the evaluation of medical countermeasures against respiratory anthrax infection.     

The critical role of pathology in the investigation of bioterrorism-related cutaneous anthrax

Cutaneous anthrax is a rare zoonotic disease in the United States. The clinical diagnosis traditionally has been established by conventional microbiological methods, such as culture and gram staining. However, these methods often yield negative results when patients have received antibiotics. During the bioterrorism event of 2001, we applied two novel immunohistochemical assays that can detect Bacillus anthracis antigens in skin biopsy samples even after prolonged antibiotic treatment. These assays provided a highly sensitive and specific method for the diagnosis of cutaneous anthrax, and were critical in the early and rapid diagnosis of 8 of 11 cases of cutaneous anthrax during the outbreak investigation. Skin biopsies were obtained from 10 of these 11 cases, and histopathological findings included various degrees of ulceration, hemorrhage, edema, coagulative necrosis, perivascular inflammation, and vasculitis. Serology was also an important investigation tool, but the results required several weeks because of the need to test paired serum specimens. Other tests, including culture, special stains, and polymerase chain reaction assay, were less valuable in the diagnosis and epidemiological investigation of these cutaneous anthrax cases. This report underscores the critical role of pathology in investigating potential bioterrorism events and in guiding epidemiological studies, a role that was clearly demonstrated in 2001 when B. anthracis spores were intentionally released through the United States postal system.     

Updating perspectives on the initiation of Bacillus anthracis growth and dissemination through its host

Since 1957, it has been proposed that the dissemination of inhalational anthrax required spores to be transported from the lumena of the lungs into the lymphatic system. In 2002, this idea was expanded to state that alveolar macrophages act as a "Trojan horse" capable of transporting spores across the lung epithelium into draining mediastinal lymph nodes. Since then, the Trojan horse model of dissemination has become the most widely cited model of inhalational infection as well as the focus of the majority of studies aiming to understand events initiating inhalational anthrax infections. However, recent observations derived from animal models of Bacillus anthracis infection are inconsistent with aspects of the Trojan horse model and imply that bacterial dissemination patterns during inhalational infection may be more similar to the cutaneous and gastrointestinal forms than previously thought. In light of these studies, it is of significant importance to reassess the mechanisms of inhalational anthrax dissemination, since it is this form of anthrax that is most lethal and of greatest concern when B. anthracis is weaponized. Here we propose a new "jailbreak" model of B. anthracis dissemination which applies to the dissemination of all common manifestations of the disease anthrax. The proposed model impacts the field by deemphasizing the role of host cells as conduits for dissemination and increasing the role of phagocytes as central players in innate defenses, while moving the focus toward interactions between B. anthracis and lymphoid and epithelial tissues.     

Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis

Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 10(5) CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores.     

Importance of srtA and srtB for growth of Bacillus anthracis in macrophages

We examined the effect of mutation of two sortase genes of Bacillus anthracis, srtA and srtB, on the ability of the bacterium to grow in J774A.1 cells, a mouse macrophage-like cell line. While disruption of either srtA or srtB had no effect on the ability of the bacteria to grow in rich culture media, mutations in each of these genes dramatically attenuated growth of the bacterium in J774A.1 cells. Complementation of the mutation restored the ability of bacteria to grow in the cells. Since the initial events in inhalation anthrax are believed to be uptake of B. anthracis spores by alveolar macrophages followed by germination of the spores and growth of the bacteria within the macrophages, these results suggest that two sortases of B. anthracis may be critical in the early stages of inhalation anthrax.     

Anthrax and carbuncle: two sides of the same coin

The disease caused by Bacillus anthracis is one of the most critical concerns to the general public and public health authorities due both to the anthrax cases caused by the intentional release of the germ in the USA at the close of 2001 when letters and packages were contaminated with anthrax spores, and the current threat of biological warfare. After a brief excursus on the history of the terms Anthrax and Carbuncle, we survey the main evidence of anthrax found in the ancient literature, and deal with the identification of the pathogenic agent responsible for the disease and the subsequent discovery of the first anthrax vaccine and its use in order to control the spread of the disease in the cattle. Finally, we examine some of the most important episodes of occupational exposure to the Bacillus anthracis that occurred in the past two centuries and the preventive measures applied both to employees and the workplace.     

Role of purine biosynthesis in Bacillus anthracis pathogenesis and virulence

Bacillus anthracis, the etiological agent of anthrax, is a spore-forming, Gram-positive bacterium and a category A biothreat agent. Screening of a library of transposon-mutagenized B. anthracis spores identified a mutant displaying an altered phenotype that harbored a mutated gene encoding the purine biosynthetic enzyme PurH. PurH is a bifunctional protein that catalyzes the final steps in the biosynthesis of the purine IMP. We constructed and characterized defined purH mutants of the virulent B. anthracis Ames strain. The virulence of the purH mutants was assessed in guinea pigs, mice, and rabbits. The spores of the purH mutants were as virulent as wild-type spores in mouse intranasal and rabbit subcutaneous infection models but were partially attenuated in a mouse intraperitoneal model. In contrast, the purH mutant spores were highly attenuated in guinea pigs regardless of the administration route. The reduced virulence in guinea pigs was not due solely to a germination defect, since both bacilli and toxins were detected in vivo, suggesting that the significant attenuation was associated with a growth defect in vivo. We hypothesize that an intact purine biosynthetic pathway is required for the virulence of B. anthracis in guinea pigs.     

应用上转磷光免疫层析技术快速定量检测炭疽芽孢

目的 建立上转磷光免疫层析技术快速定量检测炭疽芽孢。方法 利用上转磷光检测技术和免疫层析技术（双抗体夹心法）,建立检测炭疽芽孢的快速检测方法,对该方法的特异性、敏感性进行评价,在面粉、淀粉等材料中掺入炭疽芽孢模拟“白色粉末”,评价该法检测炭疽芽孢生物恐怖和环境样品的可行性,拟合检测曲线进行定量研究。结果 该法可在30min内完成定性检测,多种芽孢菌及其他病原菌评价显示该法特异性良好,检测灵敏性为104芽孢,模拟“白色粉末”的样品检测敏感性也相同,盲法考核,该法定性、定量结果较好。结论 建立的检测炭疽芽孢杆菌的上转磷光免疫层析方法,能快速、特异、灵敏地检测炭疽芽孢,适用于现场快速检测。     

Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis

Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the 2001 anthrax mailings. Most real-time PCR assays for B. anthracis have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids. In contrast, only two published chromosomal targets exist, the rpoB gene and the gyrA gene. In the present study, subtraction-hybridization with a plasmid-cured B. anthracis tester strain and a Bacillus cereus driver was used to find a unique chromosomal sequence. By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device. Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA. The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published B. anthracis Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from B. anthracis strain A2012. The importance of this chromosomal assay will be to verify the presence of B. anthracis independently of plasmid occurrence.     

Prophylaxis against anthrax

The paper presents fundamental knowledge concerning Bacillus anthracis and its potential terrorist misuse. The basic clinical forms are resumed with emphasis on inhalation infection from inspiration of B. anthracis spores. The AVA vaccine licensed in the United States, primary vaccination, protective efficacy of the vaccine, and adverse events are characterised. Stress is laid on pre-exposure and post-exposure prophylaxis of anthrax.