Studying the differential efficacy of postsymptom antitoxin treatment in type A versus type B botulism using a rabbit spirometry model.

Botulinum neurotoxin (BoNT) serotypes A, B and E are responsible for most cases of human botulism. The only approved therapy for botulism is antitoxin treatment administered to patients after symptom onset. However, a recent meta-analysis of antitoxin efficacy in human botulism cases over the past century concluded that a statistically significant reduction in mortality is associated with the use of type E and type A antitoxin, but not with type B antitoxin. Animal models could be highly valuable in studying postsymptom antitoxin efficacy (PSAE). However, the few attempts to evaluate PSAE in animals relied on subjective observations and showed ∼50% protection. Recently, we developed a novel spirometry model for the quantitative evaluation of PSAE in rabbits and used it to demonstrate full protection against BoNT/E. In the current study, a comparative evaluation of PSAE in botulism types A and B was conducted using this quantitative respiratory model. A lethal dose of each toxin induced a comparable course of disease both in terms of time to symptoms (TTS, 41.9±1.3 and 40.6±1.1 h, respectively) and of time to death (TTD, 71.3±3.1 and 66.3±1.7 h, respectively). However, in accordance with the differential serotypic PSAE observed in humans, postsymptom antitoxin treatment was fully effective only in BoNT/A-intoxicated rabbits. This serotypic divergence was reflected by a positive and statistically significant correlation between TTS and TTD in BoNT/A-intoxicated rabbits (r=0.91, P=0.0006), but not in those intoxicated with BoNT/B (r=0.06, P=0.88). The rabbit spirometry system might be useful in the evaluation toolkit of botulism therapeutics, including those under development and intended to act when antitoxin is no longer effective.

Inhalational Gentamicin Treatment Is Effective Against Pneumonic Plague in a Mouse Model.

Pneumonic plague is an infectious disease characterized by rapid and fulminant development of acute pneumonia and septicemia that results in death within days of exposure. The causative agent of pneumonic plague, Yersinia pestis (Y. pestis), is a Tier-1 bio-threat agent. Parenteral antibiotic treatment is effective when given within a narrow therapeutic window after symptom onset. However, the non-specific "flu-like" symptoms often lead to delayed diagnosis and therapy. In this study, we evaluated inhalational gentamicin therapy in an infected mouse model as a means to improve antibiotic treatment efficacy. Inhalation is an attractive route for treating lung infections. The advantages include directly dosing the main infection site, the relative accessibility for administration and the lack of extensive enzymatic drug degradation machinery. In this study, we show that inhalational gentamicin treatment administered 24 h post-infection, prior to the appearance of symptoms, protected against lethal intranasal challenge with the fully virulent Y. pestis Kimberley53 strain (Kim53). Similarly, a high survival rate was demonstrated in mice treated by inhalation with another aminoglycoside, tobramycin, for which an FDA-approved inhaled formulation is clinically available for cystic fibrosis patients. Inhalational treatment with gentamicin 48 h post-infection (to symptomatic mice) was also successful against a Y. pestis challenge dose of 10 i.n.LD50. Whole-body imaging using IVIS technology demonstrated that adding inhalational gentamicin to parenteral therapy accelerated the clearance of Y. pestis from the lungs of infected animals. This may reduce disease severity and the risk of secondary infections. In conclusion, our data suggest that inhalational therapy with aerosolized gentamicin may be an effective prophylactic treatment against pneumonic plague. We also demonstrate the benefit of combining this treatment with a conventional parenteral treatment against this rapidly progressing infectious disease. We suggest the inhalational administration route as a clinically relevant treatment modality against pneumonic plague and other respiratory bacterial pathogens.

A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.

Botulinum neurotoxins (BoNTs), the most poisonous substances known in nature, pose significant concern to health authorities. The only approved therapeutic for botulism is antitoxin. While administered to patients only after symptom onset, antitoxin efficacy is evaluated in animals mostly in relation to time postintoxication regardless of symptoms. This is most likely due to the difficulty in measuring early symptoms of botulism in animals. In this study, a rabbit spirometry model was developed to quantify early respiratory symptoms of type E botulism that were further used as a trigger for treatment. Impaired respiration, in the form of a reduced minute volume, was detected as early as 18.1 ± 2.9 h after intramuscular exposure to 2 rabbit 50% lethal doses (LD50) of BoNT serotype E (BoNT/E), preceding any visible symptoms. All rabbits treated with antitoxin immediately following symptom onset survived. Postsymptom antitoxin efficacy was further evaluated in relation to toxin and antitoxin dosages as well as delayed antitoxin administration. Our system enabled us to demonstrate, for the first time, full antitoxin protection of animals treated with antitoxin after the onset of objective and quantitative type E botulism symptoms. This model may be utilized to evaluate the efficacy of antitoxins for additional serotypes of BoNT as well as that of next-generation anti-BoNT drugs that enter affected cells and act when antitoxin is no longer effective.

Antibiotics cure anthrax in animal models.

Respiratory anthrax, in the absence of early antibiotic treatment, is a fatal disease. This study aimed to test the efficiency of antibiotic therapy in curing infected animals and those sick with anthrax. Postexposure prophylaxis (24 h postinfection [p.i.]) of guinea pigs infected intranasally with Bacillus anthracis Vollum spores with doxycycline, ofloxacin, imipenem, and gentamicin conferred protection. However, upon termination of treatment, the animals died from respiratory anthrax. Combined treatment with antibiotics and active vaccination with a protective antigen-based vaccine leads to full protection even after cessation of treatment. Delaying the initiation of antibiotic administration to over 24 h p.i. resulted in treatment of animals with anthrax exhibiting various degrees of bacteremia and toxemia. Treatment with doxycycline or ciprofloxacin cured sick guinea pigs and rabbits exhibiting bacteremia levels up to 10(5) CFU/ml. Addition of anti-protective antigen (PA) antibodies augmented the efficiency of protection, allowing the cure of guinea pigs and rabbits with 10- to 20-fold-higher bacteremia levels, up to 7 × 10(5) CFU/ml and 2 × 10(6) CFU/ml, respectively. Treatment with ciprofloxacin and a monoclonal anti-PA antibody rescued rabbits with bacteremia levels up to 4 × 10(6) CFU/ml. During antibiotic administration, all surviving animals developed a protective immune response against development of a fatal disease and subcutaneous challenge with Vollum spores. In conclusion, these results demonstrate that antibiotic treatment can prevent the development of fatal disease in respiratory-anthrax-infected animals and can cure animals after disease establishment. A therapeutic time window of 40 h to 48 h from infection to initiation of efficient antibiotic-mediated cure was observed.

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.

Immunological correlates for protection against intranasal challenge of Bacillus anthracis spores conferred by a protective antigen-based vaccine in rabbits.

Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 x 10(5) conferred protection, whereas lower titers (between 10(4) and 10(5)) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.

Postexposure prophylaxis against anthrax: evaluation of various treatment regimens in intranasally infected guinea pigs.

The efficiency of postexposure prophylaxis against Bacillus anthracis infection was tested in guinea pigs infected intranasally with either Vollum or strain ATCC 6605 spores (75 times the 50% lethal dose [LD(50)] and 87 times LD(50,) respectively). Starting 24 h postinfection, animals were treated three times per day for 14 days with ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole (TMP-SMX). Administration of cefazolin and TMP-SMX failed to protect the animals, while ciprofloxacin, tetracycline, and erythromycin prevented death. Upon cessation of treatment all erythromycin-treated animals died; of the tetracycline-treated animals, two of eight infected with Vollum and one of nine infected with ATCC 6605 survived; and of the ciprofloxacin group injected with either 10 or 20 mg/kg of body weight, five of nine and five of five animals, respectively, survived. To test the added value of extending the treatment period, Vollum-infected (46 times the LD(50)) animals were treated for 30 days with ciprofloxacin or tetracycline, resulting in protection of eight of nine and nine of nine animals, respectively. Once treatment was discontinued, only four of eight and five of nine animals, respectively, survived. Following rechallenge (intramuscularly) of the survivors with 30 times the LD(50) of Vollum spores, all ciprofloxacin-treated animals were protected while none of the tetracycline-treated animals survived. In an attempt to confer protective immunity lasting beyond the termination of antibiotic administration, Vollum-infected animals were immunized with a protective antigen (PA)-based vaccine concurrently with treatment with either ciprofloxacin or tetracycline. The combined treatment protected eight of eight and nine of nine animals. Following cessation of antibiotic administration seven of eight and eight of eight animals survived, of which six of seven and eight of eight resisted rechallenge. These results indicate that a combined treatment of antibiotics together with a PA-based vaccine could provide long-term protection to prevent reoccurrence of anthrax disease.