Potential Biological Warfare Agents Research Articles

A terbium-functionalized ZIF-8 nanosensor for rapid and sensitive detection of an anthrax spore biomarker

Because anthrax spores are highly lethal to humans and animals as well as potential biological warfare agents, there is a great need for rapid, sensitive, and selective quantification of dipicolinic acid (DPA) as a biomarker for anthrax spores.

Melioidosis: Is it Still Underdiagnosed?

Abstract Burkholderia pseudomallei, a Gram-negative bacterium, is the cause of Melioidosis, a disease that is known to cause significant morbidity and mortality in Southeast Asia. It is also classified as a potential biological warfare agent and is considered a category B priority pathogen. Despite being common in these areas, melioidosis is often underdiagnosed due to lack of awareness of its clinical and microbiological characteristics, limited health-care resources, and the varied ways in which it can present. It is important to recognize the clinical features of melioidosis early on and properly manage the disease to reduce its impact.

Rapid and sensitive detection of botulinum toxin type A in complex sample matrices by AlphaLISA.

Botulinum toxin A(BoNT/A) is a neurotoxin produced by the bacteria Clostridium botulinum, which can cause serious food poisoning and is recognized as a potential biological warfare agent. BoNT/A is does not degrade easily and can remain in the complex matrix for a long time. Meanwhile, the poisonous dose of botulinum toxin exceptionally low and intravenous human lethal doses estimated at 1-3 ng/kg. Therefore, sensitive and accurate detection methods suitable for testing a wide range of complex samples are urgently needed. To this end, the "amplified luminescent proximity homogeneous assay linked immunosorbent assay" (AlphaLISA) was established for the detection of BoNT/A and its detection efficacy in plasma, beverage, food, and other complex samples was evaluated. The results showed that this method can very effectively resist matrix interference. The detection time is rapid, reaching a detection limit for all samples of up to 0.1 ng/mL in only 30 min. BoNT/A can also be accurately detected in vomit samples of patients with clinical food poisoning. This study demonstrates that AlphaLISA is an effective tool for the detection of BoNT/A in complex samples and can potentially be developed for commercial use in the future.

Safety and immunogenicity of a trivalent virus-like particle vaccine against western, eastern, and Venezuelan equine encephalitis viruses: a phase 1, open-label, dose-escalation, randomised clinical trial

Western (WEEV), eastern (EEEV), and Venezuelan (VEEV) equine encephalitis viruses are mosquito-borne pathogens classified as potential biological warfare agents for which there are currently no approved human vaccines or therapies. We aimed to evaluate the safety, tolerability, and immunogenicity of an investigational trivalent virus-like particle (VLP) vaccine, western, eastern, and Venezuelan equine encephalitis (WEVEE) VLP, composed of WEEV, EEEV, and VEEV VLPs. The WEVEE VLP vaccine was evaluated in a phase 1, randomised, open-label, dose-escalation trial at the Hope Clinic of the Emory Vaccine Center at Emory University, Atlanta, GA, USA. Eligible participants were healthy adults aged 18-50 years with no previous vaccination history with an investigational alphavirus vaccine. Participants were assigned to a dose group of 6 μg, 30 μg, or 60 μg vaccine product and were randomly assigned (1:1) to receive the WEVEE VLP vaccine with or without aluminium hydroxide suspension (alum) adjuvant by intramuscular injection at study day 0 and at week 8. The primary outcomes were the safety and tolerability of the vaccine (assessed in all participants who received at least one administration of study product) and the secondary outcome was immune response measured as neutralising titres by plaque reduction neutralisation test (PRNT) 4 weeks after the second vaccination. This trial is registered at ClinicalTrials.gov, NCT03879603. Between April 2, 2019, and June 13, 2019, 30 trial participants were enrolled (mean age 32 years, range 21-48; 16 [53%] female participants and 14 [47%] male participants). Six groups of five participants each received 6 μg, 30 μg, or 60 μg vaccine doses with or without adjuvant, and all 30 participants completed study follow-up. Vaccinations were safe and well tolerated. The most frequently reported symptoms were mild injection-site pain and tenderness (22 [73%] of 30) and malaise (15 [50%] of 30). Dose-dependent differences in the frequency of pain and tenderness were found between the 6 μg, 30 μg, and 60 μg groups (p=0·0217). No significant differences were observed between dosing groups for any other reactogenicity symptom. Two adverse events (mild elevated blood pressure and moderate asymptomatic neutropenia) were assessed as possibly related to the study product in one trial participant (60 μg dose with alum); both resolved without clinical sequelae. 4 weeks after second vaccine administration, neutralising antibodies were induced in all study groups with the highest response seen against all three vaccine antigens in the 30 μg plus alum group (PRNT80 geometric mean titre for EEEV 60·8, 95% CI 29·9-124·0; for VEEV 111·5, 49·8-249·8; and for WEEV 187·9, 90·0-392·2). Finally, 4 weeks after second vaccine administration, for all doses, the majority of trial participants developed an immune response to all three vaccine components (24 [83%] of 29 for EEEV; 26 [90%] of 29 for VEEV; 27 [93%] of 29 for WEEV; and 22 [76%] of 29 for EEEV, VEEV, and WEEV combined). The favourable safety profile and neutralising antibody responses, along with pressing public health need, support further evaluation of the WEVEE VLP vaccine in advanced-phase clinical trials. The Vaccine Research Center of the National Institute of Allergy and Infectious Diseases, National Institutes of Health funded the clinical trial. The US Department of Defense contributed funding for manufacturing of the study product.

Characterization of immunoglobulin and cytokine responses in Burkholderia mallei infected equids

Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aerosol transmission, lack of effective vaccine and treatment make this organism a potential agent of biological warfare. Results of experimental B. mallei infection in mouse and non-human primates and immunization with live attenuated B. mallei strains demonstrated that activation of early innate and adaptive immune responses play a critical role in controlling B. mallei infection. However, the immune response elicited by the primary hosts (equids) B. mallei infection is poorly understood. Therefore, we aimed to investigate immune responses in glanders affected horses (n = 23) and mules (n = 1). In this study, chronically infected equids showed strong humoral responses (IgM, IgG and IgA) specific to B. mallei type 6 secretory proteins such as Hcp1, TssA and TssB. The infected equids also elicited robust cellular responses characterized by significantly elevated levels of IFN-γ, TNF-α, IL-12, IL-17 and IL-6 in PBMCs. In addition, stimulation of equine PBMCs by Hcp1 resulted in the further elevation of these cytokines. Thus, the present study indicated that antibody response and T helper cell (Th) type 1-associated cytokines were the salient features of chronic B. mallei infection in horses. The immune responses also suggest further evaluation of these proteins as potential vaccine candidates.

Cellular and molecular level host-pathogen interactions in Francisella tularensis: A microbial gene network study

Due to the high infectivity and fatal effect on human population, Francisella tularensis (F. tularensis) is classified as a potential biological warfare agent. The interaction between host and pathogen behind the successful establishment of F. tularensis infection within the human host is largely unknown. In our present work, we have studied the molecular level interactions between the host cellular components and F. tularensis genes to understand the interplay between the host and pathogen. Interestingly, we have identified the pathways associated with the pathogen offensive strategies that help in invasion of host defensive systems. The F. tularensis genes purL, katG, proS, rpoB and fusA have displayed high number of interactions with the host genes and thus play a crucial role in vital pathogen pathways. The pathways identified were involved in adaptation to different stress conditions within the host and might be crucial for designing new therapeutic interventions against tularemia.

Synthesis and Decontamination Effect on Chemical and Biological Agents of Benzoxonium-Like Salts.

Benzoxonium chloride belongs to the group of quaternary ammonium salts, which have been widely used for decades as disinfectants because of their high efficacy, low toxicity, and thermal stability. In this study, we have prepared the C10-C18 set of benzoxonium-like salts to evaluate the effect of their chemical and biological decontamination capabilities. In particular, biocidal activity against a panel of bacterial strains including Staphylococcus aureus in biofilm form was screened. In addition, the most promising compounds were successfully tested against Francisella tularensis as a representative of potential biological warfare agents. From a point of view of chemical warfare protection, the efficiency of BOC-like compounds to degrade the organophosphate simulant fenitrothion was examined. Notwithstanding that no single compound with universal effectiveness was identified, a mixture of only two compounds from this group would be able to satisfactorily cover the proposed decontamination spectrum. In addition, the compounds were evaluated for their cytotoxicity as a basic safety parameter for potential use in practice. In summary, the dual effect on chemical and biological agents of benzoxonium-like salts offer attractive potential as active components of decontamination mixtures in the case of a terrorist threat or chemical or biological accidents.

Eight Years of Collaboration on Biosafety and Biosecurity Issues Between Kazakhstan and Germany as Part of the German Biosecurity Programme and the G7 Global Partnership Against the Spread of Weapons and Materials of Mass Destruction

In 2013, the German Federal Foreign Office launched the German Biosecurity Programme with the aim to minimise risks associated with biological substances and pathogens. In this context, the German-Kazakh Network for Biosafety and Biosecurity was established in 2013 and constitutes a successful collaboration between Kazakh and German biomedical organisations, under the co-management of the Bundeswehr Institute of Microbiology (IMB), and the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH. Ever since then, a network of scientists, stake holders and policymakers has been established, aiming to work on highly pathogenic, potential biological warfare agents with the focus on biosafety and biosecurity, surveillance, detection and diagnostics, networking and awareness raising of these agents in Kazakhstan. Over the past 8 years, the project members trained four PhD candidates, organised over 30 workshops and trainings with more than 250 participants and conducted more than 5,000 PCR assays and 5,000 serological analyses for surveillance. A great success was the description of new endemic areas for Orthohantaviruses, the mixture of two Crimean-Congo haemorrhagic fever virus genetic clusters, new foci and genetic information on tick-borne encephalitis virus and rickettsiae in Kazakh oblasts. The latter even led to the description of two new genogroups. Furthermore, joint contributions to international conferences were made. In this report, we summarise the evolution of the German-Kazakh Network for Biosafety and Biosecurity and critically reflect on the strengths and possible weaknesses. We were able to establish a viable network of biosafety and biosecurity shareholders and to accomplish the aims of the German Biosecurity Programme to lower biosecurity risks by increased awareness, improved detection and diagnostic methods and surveillance. Further, we reflect on forthcoming aspects to lead this interstate endeavour into a sustainable future.

A comprehensive review on avian chlamydiosis: a neglected zoonotic disease.

Avian chlamydiosis is one of the important neglected diseases with critical zoonotic potential. Chlamydia psittaci, the causative agent, affects most categories of birds, livestock, companion animals, and humans. It has many obscured characters and epidemiological dimensions, which makes it unique among other bacterial agents. Recent reports on transmission from equine to humans alarmed the public health authorities, and it necessitates the importance of routine screening of this infectious disease. High prevalence of spill-over infection in equines was associated with reproductive losses. Newer avian chlamydial species are being reported in the recent years. It is a potential biological warfare agent and the disease is an occupational hazard mainly to custom officers handling exotic birds. Prevalence of the disease in wild birds, pet birds, and poultry causes economic losses to the poultry industry and the pet bird trade. Interestingly, there are speculations on the ‘legal’ and ‘illegal’ bird trade that may be the global source of some of the most virulent strains of this pathogen. The mortality rate generally ranges from 5 to 40% in untreated cases, but it can sometimes be higher in co-infection. The intracellular lifestyle of this pathogen makes the diagnosis more complicated and there is also lack of accurate diagnostics. Resistance to antibiotics is reported only in some pathogens of the Chlamydiaceae family, but routine screening may assess the actual situation in all pathogens. Due to the diverse nature of the pathogen, the organism necessitates the One Health partnerships to have complete understanding. The present review focuses on the zoonotic aspects of avian chlamydiosis with its new insights into the pathogenesis, transmission, treatment, prevention, and control strategies. The review also briefs on the basic understandings and complex epidemiology of avian chlamydiosis, highlighting the need for research on emerging one health perspectives.

Personalized Production, Non-Genomic Engineering, and Time-Resolved Proteomics of Therapeutic Phages for Biohazardous and Multidrug-Resistant Bacteria

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is currently hampered by a lack of rapid phage production and efficient bioengineering and characterization capabilities. Thus, we have developed a comprehensive cell-free platform for time-efficient and personalized manufacturing, transient bioengineering, and proteomic characterization of a broad spectrum of phages. We validated 40 hypothetical proteins, including non-structural proteins, from T7 and CLB-P3 phages using high-resolution mass spectrometry and identified a new late-expressing gene cluster. Just a few microliters of the one-pot reaction produce effective doses of phages against enteroaggregative E. coli (EAEC) and potential biological warfare agents such as Yersinia pestis. By coexpressing suitable host factors, we could extend the range of cell-free phage production to include selective phages targeting gram-positive bacteria. We also introduce a transient, non-genomic phage engineering method, which adds functionalities for only one replication cycle. Finally, we showcase a pipeline for personalized phage therapy of a multidrug-resistant ESKAPE pathogen using a newly isolated and in-vitro produced phage against Klebsiella pneumoniae. We expect our cell-free methodology to enable accelerated reverse and forward phage engineering and the safe and customized production of clinical-grade therapeutic bacteriophages.

Development of novel electrochemical sensor for the detection of biological warfare agents: enzyme, antibody, and DNA free

There is an urgent need to develop a novel sensor that can quickly and accurately detect biological warfare agents (BWAs). A novel electrochemical sensor was specially designed and developed for Staphylococcus aureus as a potential BWAs without using any enzyme, antibody, or genetic material. Cyclic voltammetry (CV), linear resistance polarization (LRP), chronoamperometry, impedance spectroscopy, and Differential Pulse Voltammetry (DPV) techniques were used for fast and accurate detection of BWAs. Co-precipitation technique was used for the synthesis of CuO nanoparticles (NPs) for their use in the electrochemical sensor. The effect of temperature, stirring and addition of CuO NPs was thoroughly investigated. The storage, stability, and performance of electrochemical sensor was tested for 45 days. The current response of the electrochemical sensor was found linear with the correlation coefficient, R2 = 0.991. The major advantage of this novel electrochemical sensor is that immobilization of enzyme, antibody, or DNA is not required in sensing technology as the immobilization process makes biosensors a highly specific. The novel electrochemical sensor was very quick, accurate, non-specific, and provide reliable results in the qualitative and quantitative estimation of pathogenic bacteria as a potential BWAs. A novel electrochemical sensor was specially designed and developed for the detection of Staphylococcus aureus pathogenic bacteria as a potential BWAs without using any enzyme, antibody, or genetic material. Cyclic voltammetry, linear resistance polarization, chronoamperometry, impedance spectroscopy, and Differential Pulse Voltammetry (DPV) experiments were carried out for fast and accurate detection of BWAs. Storage, stability, and reusability of the electrochemical sensor was tested for continuous 45 days. The current response of the electrochemical sensor was found to be linear with the correlation coefficient, R2 = 0.991. The major advantage of this novel sensor is that immobilization of enzyme, antibody, or DNA is not required in sensing technology as the immobilization process makes biosensor a highly specific. The novel electrochemical sensor was proved to be very quick, accurate, non-specific, and provide reliable results in the qualitative and quantitative estimation of pathogenic bacteria as a potential BWAs.

Tularemia: a re-emerging tick-borne infectious disease.

Tularemia is a bacterial disease of humans, wild, and domestic animals. Francisella tularensis, which is a Gram-negative coccobacillus-shaped bacterium, is the causative agent of tularemia. Recently, an increase in the number of human tularemia cases has been noticed in several countries around the world. It has been reported mostly from North America, several Scandinavian countries, and certain Asian countries. The disease spreads through vectors such as mosquitoes, horseflies, deer flies, and ticks. Humans can acquire the disease through direct contact of sick animals, consumption of infected animals, drinking or direct contact of contaminated water, and inhalation of bacteria-loaded aerosols. Low infectious dose, aerosol route of infection, and its ability to induce fatal disease make it a potential agent of biological warfare. Tularemia leads to several clinical forms, such as glandular, ulceroglandular, oculoglandular, oropharyngeal, respiratory, and typhoidal forms. The disease is diagnosed through the use of culture, serology, or molecular methods. Quinolones, tetracyclines, or aminoglycosides are frequently used in the treatment of tularemia. No licensed vaccine is available in the prophylaxis of tularemia and this is need of the time and high-priority research area. This review mostly focuses on general features, importance, current status, and preventive measures of this disease.

Cu(II)-assisted orange/green dual-emissive carbon dots for the detection and imaging of anthrax biomarker.

The spores of Bacillus anthracis are highly deadly to human beings and animals, and are concurrently potential biological warfare agents. Hence, the rapid and sensitive monitoring Bacillus anthracis biomarker, dipicolinic acid (DPA), is very desirable. Herein, orange/green dual-emissive carbon dots (OG-CDs) were synthesized via the hydrothermal approach. The OG-CDs not only emitted dual fluorescence at 527 and 590nm under the single 503nm excitation, but also exhibited excellent water solubility, good photostability and great salt tolerance. The fluorescence of the OG-CDs at 527nm can be completely quenched when chelated with Cu(II). However, because of the stronger chelation between DPA and Cu(II), the fluorescence restored rapidly on subsequent addition of DPA. As such, the CD-Cu(II) system can be used for determination of DPA based on the fluorescence "off-on" response. Under optimum conditions, the detection limit for DPA was 56nM, with a linear range of 0.5-12.5μM. The established CD-Cu(II) based spectrofluorometric method has been applied to the analysis of DPA in real water samples with recoveries of 93.6%-104.3%. More remarkably, the CD-Cu(II) probe also has been successfully applied for the imaging of DPA in Escherichia coli with excellent bio-compatibility.

Putative serum protein biomarkers for epsilon toxin exposure in mouse model using LC-MS/MS analysis

Epsilon toxin (ETX), produced by Clostridium perfringens Type B or type D strains, is a potential biological and toxin warfare (BTW) agent, largely for its very high toxicity. The toxin is implicated in several animal diseases. Using LC-MS/MS analysis, we report here elucidation of putative serum maker proteins for ETX exposure with an objective of the early diagnosis of intoxication. Of 166 consensus proteins (488 peptides), showing ETX-induced alterations, 119 proteins exhibited increase and 47 proteins showed decreased abundance in serum, as revealed by SWATH (DIA) acquisition on LC-MS/MS and label free quantitative analysis of control and test samples. Complement and coagulation cascade, nitrogen metabolism, negative regulation of peptidase activity, and response to ROS were among the biological processes and pathways perturbed by the ETX exposure. Interaction network indicated enzyme inhibitor activity, detoxification of ROS, and steroid binding functions were the major interaction networks for the proteins with increased abundance, while, hemostasis and structural molecule activity were the prominent networks for the down-regulated proteins. Validation studies were carried out by immunoprecipitation, ELISA, and Western blot analysis of selected proteins to demonstrate diagnostic potential of the putative marker proteins of ETX exposure.

Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6pgmL-1 with a large dynamic range from 10 pgmL-1 to 1 µgmL-1.

Glanders: re-emergence of an ancient zoonosis

Glanders, although known to be endemic in certain regions/countries of the Old and New Worlds for centuries, had been largely overlooked as a threat to equine and human health until the disease re-emerged in the Middle East in 2004. The exponential growth in international horse movements, both legal and illegal, mainly for performance purposes, has enhanced the risk of global spread of glanders in the Middle East and elsewhere. Ever since the First World War, the glanders bacillus has been recognised as a potential biological warfare agent.

A real-time loop mediated isothermal amplification assay for molecular detection of Burkholderia mallei, the aetiological agent of a zoonotic and re-emerging disease glanders

Burkholderia mallei, a potential biological warfare agent, is the causative agent of an infectious, fatal, and zoonotic disease, called glanders. Accurate and early diagnosis of glanders is important to control the disease lethality and infection spread. Molecular detection of B. mallei is considered strenuous because B. mallei is a subtractive genomic clone of B. pseudomallei. The present study was aimed at development of a real-time LAMP assay for detection of B. mallei. The LAMP assay was highly sensitive and could detect ≥250 fg of genomic DNA of B. mallei and ≥100 copies of recombinant plasmid containing target DNA sequence. In artificially spiked blood and water samples, it could detect ≥2.1 × 103 and ≥4.73 × 102 CFU/mL of B. mallei, respectively. The assay was highly specific for B. mallei as none of the other bacteria used in the study tested positive. The reported LAMP assay being simple and rapid can be a viable alternative to PCR-based glanders diagnostic assays in glanders endemic regions with resource-limited settings.

A ratiometric fluorescent probe for sensitive detection of anthrax biomarker based on terbium-covalent organic polymer systems

Bacillus anthrax is harmful to humans and animals and can be used as potential biological warfare agents. Dipicolinic acid (DPA) is usually employed to detect Bacillus anthrax as an important biomarker, therefore, it is of great significance to develop highly selective and sensitive DPA quantitative methods. Herein, terbium-covalent organic polymer (Tb-COP) as a ratiometric fluorescent probe was synthesized and applied to quantitatively detect DPA. In the probe, COP played a role of fluorescent reference, while Tb acted as specific recognition element and response signal. When DPA was introduced to Tb-COP, DPA replaced water molecule to coordinate with Tb3+ and transferred the absorbed energy to it, resulting in an "antenna effect". The approach exhibited wide linear range from 0.1 to 30 μM and low detection limit of 13.5 nM. It was the first time that Tb-COP was used as a ratiometric fluorescent probe for DPA sensing, which provided a non-interference, self-calibration, and effective method for sensitive and selective detection of DPA.

A highly selective lanthanide-containing probe for ratiometric luminescence detection of an anthrax biomarker.

Since anthrax spores are highly lethal to human beings and animals, and also potential biological warfare agents, it is highly desirable to develop simple, robust and easy-device sensors that are rapid and sensitive, and can selectively detect the Bacillus anthrax biomarker dipicolinic acid (DPA) quantitatively. Herein, we report a ratiometric luminescent "turn-on" sensor via mixing Eu3+ ions, sodium polyacrylate and internal luminescence reference in water, which can identify the Bacillus anthrax biomarker dipicolinic acid (DPA) quantitatively with outstanding selectivity over aromatic ligands and amino acids, even including all of the 5 DPA isomers.

Molecular modeling studies on the interactions of aflatoxin B1 and its metabolites with the peripheral anionic site of human acetylcholinesterase

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus. Among them, aflatoxin B1 (AFB1) is the most frequent type in nature and the most carcinogenic for mammals. It can contaminate many kinds of food like seeds, oil, olives, milk, dairy products, corn and meat, causing acute and chronic damages to the organism, especially in the liver, being, for this reason, considered highly hepatotoxic. AFB1 is also a mixed inhibitor of the enzyme acetylcholinesterase (AChE). This fact, together with its high toxicity and carcinogenicity, turns AFB1 into a potential chemical and biological warfare agent, as well as its metabolites. In order to investigate this, we performed inedited molecular modeling studies on the interactions of AFB1 and its metabolites inside the peripheral anionic site of human AChE (HssAChE), to verify their stability, suggest the preferential ways of inhibition, and compare their behavior to each other. Our results suggest that all metabolites can be better inhibitors of HssAChE than AFB1 and that AFBO and AFM1, the most toxic and carcinogenic metabolites of AFB1, are also the most effective HssAChE inhibitors among the AFB1 metabolites.Communicated by Ramaswamy H. Sarma