Abstract
Biomonitoring of food and environmental matrices is critical for the rapid and sensitive diagnosis, treatment, and prevention of diseases caused by toxins. The U.S. Centers for Disease Control and Prevention (CDC) has noted that toxins from bacteria, fungi, algae, and plants present an ongoing public health threat, especially since some of these toxins could compromise security of the food supply. Botulinum neurotoxins (BoNTs), produced by Clostridium spp., are among those bacterial toxins that pose life-threatening danger to humans. BoNTs inhibit the release of acetylcholine at peripheral cholinergic nerve terminals and cause flaccid paralysis. BoNTs are grouped in seven serotypes and many subtypes within these groups. Rapid and accurate identification of these toxins in contaminated food as well as in environmental matrices can help direct treatment. Herein, we discuss current methods to detect BoNTs with a focus on how these technologies have been used to identify toxins in various food and environmental matrices. We also discuss the emergence of new serotypes and subtypes of BoNTs and the increasing number of cases of botulism in wildlife. Finally, we consider how environmental changes impact food safety for humans and present new challenges for detection technology.
Keywords
- Botulism
- Toxins
- Food matrix
- Environmental detection
- Foodborne illness
1. Introduction
The U.S. Centers for Disease Control (CDC) have summarized the risks that biological toxins pose to human health [1]. Bacteria, fungi, parasites, and plants all produce toxins in the environment that can impact food safety. Furthermore, changes in the environment have caused emergence of new problems associated with toxins. One example is the production of toxins by
Strains in Group II are classified as “non-proteolytic” [5, 6]. These
Group III botulinum produces toxins of serotype C or D and is associated with avian and non-human mammalian botulism [5, 6]. Whole genome sequencing analysis indicates that strains of physiological group III are probably more related to
Bacteriophages contain the neurotoxin genes of
At the amino acid sequence level, BoNT serotypes can differ from one other by 34–64% [5, 6]. Significant genetic variation within each serotype has also been observed. In fact, 32 toxin subtypes with amino acid sequence differences of 2.6–32% have been identified thus far, and more will likely be identified in the future [5, 6]. This serotype and subtype diversity confound direct antibody and molecular-based assay designs. It is rare that one probe can bind to all serotypes. In
2. Important factors to consider when developing toxin detection assays
The development of a robust assay for the detection of any pathogen or biological product of a pathogen (such as a toxin) requires consideration of several factors: sensitivity, specificity, matrix effects, and biological activity [8–10]. Each of these factors is briefly discussed below in the context of assay methods for
3. The mouse bioassay
In the laboratory, a rodent bioassay is considered the “gold standard” method for detecting BoNTs [8–10]. Despite much effort to replace the use of animals, it is still the most sensitive and reliable assay to model all aspects of BoNT intoxication: binding, translocation, enzymatic activity, and pathology. Alternatives to the mouse bioassay have been developed (discussed below) with shorter assay times and equal or greater sensitivity.
The mouse assay quantitatively determines the amount of BoNT required to kill all mice in a test group. This measurement is expressed as a minimal lethal dose (MLD). Although protocols may vary, mice are usually injected intraperitoneally with 0.5 mL of BoNT sample in a dilution series and then monitored over several days for signs of intoxication and death [11, 12]. If enough sample is available for an assay, the specific neurotoxin can be identified using neutralization with antibodies specific to each of the neurotoxin serotypes (A–G). The toxin serotype is therefore revealed based on which antibody confers protection from death. The mouse bioassay is highly sensitive and useful for detection of different neurotoxins in different matrices. However, despite its versatility, the mouse assay has limitations that include: long assay times and the use of animals requiring specialized animal facilities, substantial costs, trained staff, and consideration of ethical issues, especially when death is used as an endpoint. There is also substantial variation in results observed among different research laboratories [8–12].
Alternative “refined” animal assays that do not use death as an endpoint, such as the mouse phrenic nerve hemi-diaphragm assay, have been evaluated [13, 14]. Despite being more sensitive and rapid compared to the use of whole animals, these assays usually require the use of specific equipment and personnel with specialized training. Furthermore, these alternative animal assays are not feasible with larger samples and those containing a complex matrix. However, a recent study described an
4. DNA and other nucleic acid–based methods
Numerous nucleic acid methods have been developed for detecting clostridial DNA in biological and environmental matrices. The polymerase chain reaction (PCR) to identify the presence of
Multiplex PCR methods have also been developed to analyze unknowns for a battery of different targets such as different pathogens and/or associated gene products of these pathogens. Multiplexed assays employ different combinations or sets of PCR primers, each one specific for a gene of interest, to amplify multiple targets in one PCR tube. One such multiplex method could possibly discriminate among BoNT serotypes A, B, E, and F. As previously described, Peck et al. [16] developed a culture enrichment method that, when coupled with multiplex PCR, could identify strains of
Real-time PCR (RT-PCR) or quantitative PCR (qPCR) is also useful in studies of gene expression, specifically differential expression of genes under various environmental conditions or comparative studies of different organisms. For detection of clostridial DNA, RT-PCR methods examine expression of the NTNH (non-toxic, non-hemagglutinin) and numerous other genes in
The GeneDisc Cycler is an apparatus to perform RT-PCR applications using the GeneDisc system. The GeneDisc is a disposable plastic reaction tray that is the size of a compact disc. This method has been designed for simultaneously testing for the BoNT/A, BoNT/B, BoNT/E, and BoNT/F genes. In 2011, Fach et al. evaluated the GeneDisc Cycler equipment with neurotoxin-producing clostridia and non-BoNT-producing bacteria isolated from various clinical, food, and environmental samples [20]. Results obtained using this “macroarray” were also compared to the mouse bioassay. The toxin genes were detected in all clostridial serotypes A, B, E, and F as well as in toxigenic
Recently, Kolesnikov et al. [22] described a new method called “proteolytic PCR” in which PCR is used to assay the proteolytic activity of botulinum toxin. This technology starts with DNA–protein complexes attached to a solid phase. Proteolytic cleavage releases DNA into solution. The DNA can then serve as a template for PCR. This study described its use to detect botulinum toxin and tetanus toxin proteolytic activity [22].
5. Immunological and antibody-based assays
Enzyme-linked immunosorbent assay (ELISA) is a common assay used to detect BoNTs. This method utilizes anti-BoNT capture and detector antibodies arranged in a “sandwich” format. The detection formats are most commonly luminescent- or colorimetric-based. Prior generations of BoNT immunoassays were approximately 10 times less sensitive than the mouse bioassay described in the previous section. Although not as sensitive, ELISA methods are relatively fast, inexpensive, and simple to perform. They are also less subject to inhibitory matrix effects. An amplified ELISA for detecting toxins in food matrices has also been described [23]. Toxins for serotypes A, B, E, and F could be detected in liquids, solid food, and semisolid food. Assay performance was evaluated in a range of food matrices, such as broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meats, and dairy foods. The assay sensitivity varied for each botulinum serotype. The tests readily detected 2 ng/mL of serotypes A, B, E, and F in various foods tested. Recently, traditionally formatted, very sensitive sandwich ELISAs used high affinity mAbs against BoNT/A and BoNT/B to detect BoNT/A as low as 5 and 25 pg/mL in buffer and in a milk matrix, respectively; and BoNT/B at 100 fg/mL and 39 pg/mL in buffer and milk matrix, respectively [24–26].
These mAbs were used in an electrochemiluminesence (ECL) immunosorbent assay using the Sector 2400 Imager (Meso Scale Discovery [MSD], Rockville, MD, USA) instrument [27, 28]. Detection sensitivities for BoNT/A in this system were similar to traditional ELISAs in buffer but were markedly improved in liquid food matrices because of the reduced background signal. The higher sensitivity and reduced time required for these new immunosorbent methods make them potential alternatives to the mouse bioassay. Sharma et al. recently developed another ECL assay for simultaneous detection of several biothreat agents (including clostridial neurotoxins) in milk products, with limit of detection (LOD) of 40 pg/mL for BoNT/A complex [28]. The ECL assay was also successfully applied to screen
Cheng and Stanker [27] evaluated the performance of antitoxin mAbs using the same electrochemiluminescence immunoassay platform (Sector 2400 Imager, MSD). The ELISA and ECL methods were observed to be more sensitive than the mouse bioassay. In fact, the ECL assay was able to outperform ELISA in terms of detection sensitivity—including food matrices spiked with BoNT/A and in some food matrices spiked with BoNT/B. The ELISA and ECL methods are fast and simple alternatives to the mouse bioassay and can be used for detecting BoNTs in food matrices and serum samples.
One example of mAb development using a recombinant immunogen was the work of Liu et al. [29], who expressed the recombinant H(C) subunit of BoNT type A (rAH(C)). Two out of 56 mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA) with LOD for both rAH(C) and BoNT/A of 0.45 pg/mL. This CLEIA can be used to detect BoNT/A in matrices, such as milk and beef extract. This method is 20–40-fold more sensitive than the mouse bioassay and takes only 3 hours to complete, making it a useful method to detect and quantify BoNT/A.
The multiplex technology discussed above to detect nucleic acid has also been applied to the development of methods to analyze multiple epitopes on a single antigen and multiple targets in a single sample. This approach uses multiple mAbs as well as polyclonal antibodies to reduce false-positive and false-negative results. A commercialized system, Luminex xMAP technology, utilizes microsphere beads conjugated with antibodies. It employs paramagnetic beads instead of non-magnetic polystyrene beads and is very useful for the analysis of food matrices. The antibody-bead complexes detect multiple epitopes in a single sample. This technology was used to detect abrin, ricin, BoNTs, and staphylococcal enterotoxins in spiked food samples [30].
Zhang et al. [31] developed ELISA-based protein antibody microarrays to simultaneously detect six serotypes of BoNTs. Using numerous different food and other matrices, the microarray is capable of detecting BoNT serotypes A through F. Using engineered, high-affinity antibodies, these serotypes were detected to similar levels in various matrices and were comparable to detection in buffer.
Accurate and sensitive detection of contaminated food and other biological samples in the environment is critical. Brunt et al. [32] have developed an affinity column-based assay for detecting neurotoxin in food matrices—specifically serotypes A, B, E, and F. The detection limit for BoNT/A was reported as 0.5 ng, which is two-fold more sensitive than lateral flow methods (also see Section 6) [32]. For serotypes B, E, and F, the minimum detection limit ranged from 5 to 50 ng. Although not as sensitive as ELISA or mouse bioassay, rapid immunochromatographic methods generally require only 15–30 minutes to complete. They do not require enrichment steps and are amenable to use in the field.
Koh et al. have presented a new technology called SpinDx [33]. This method utilizes a centrifugal microfluidic platform to detect BoNTs based on a sedimentation immunoassay. A reagent mixture is prepared, consisting of capture beads conjugated with target-specific antibodies and fluorescent detection antibodies. The reagents are mixed with the sample and forced through a channel containing dense medium, a process that washes the sample and removes interfering substances. The beads that collect at the end of the channel are queried to determine the amount of antigen bound. SpinDx was used to quantify BoNTs with sensitivity that surpassed the mouse assay.
6. Lateral flow methods
The development of lateral flow methods for detecting toxins has also led to the commercial availability of numerous kits for sensitive and rapid testing. Lateral flow methods employ capture antibodies that are “printed” on nitrocellulose membranes in a process akin to inkjet printing technology. Detection antibodies are labeled with visible materials, such as colloidal gold or colored latex beads. The sample is added to a reagent pad containing labeled toxin-binding detection antibodies and is wicked across the membrane. Toxin is retained by the capture antibody, which also concentrates the labeled detection antibody. A positive reaction is revealed as a colorimetric change and is presented as a line on the device. In general, lateral flow methods are qualitative and simply determine the presence or absence of neurotoxin. Sharma et al. [34] compared several commercial lateral flow devices for their capacities to detect toxin in food samples. They were able to detect BoNT/A and BoNT/B as low as 10 ng/mL and BoNT/E at 20 ng/mL in various liquids, such as milk, soft drinks, and fruit juices. Ching et al. [35] used the same mAbs described in the ELISA section above [24–26] in lateral flow devices to achieve sensitivities of 0.5 and 1 ng/mL for BoNT/A in buffer and milk, respectively. Although simple lateral flow tests have lower sensitivities compared to other methods, they produce rapid results and are most useful for the rapid screening of samples suspected of frequent contamination at relatively high level of BoNT. They have many applications and are ideal for field use by non-technical personnel. Self-contained and not necessarily requiring additional reagents or equipment, they can be easily interpreted in the field.
An innovative approach for toxin detection has recently been developed that combines antibodies with the amplification power of PCR, immuno-PCR (I-PCR) [36]. In I-PCR, template DNA is conjugated to the antibody, replacing a secondary antibody conjugated to the detection enzyme. Upon binding of toxin by the antibody, the presence of toxin is revealed using PCR. Chao et al. [36] described an I-PCR method for detection of BoNT/A with femtogram (10−15 g) sensitivity. These investigators compared competitive and sandwich ELISA to the I-PCR method. The I-PCR method was 103–105 times more sensitive with LODs for the ELISA methods of about 50 fg. The use of I-PCR for highly sensitive detection of BoNT in food matrices or other biological and environmental backgrounds has yet to be reported (as of late 2015).
7. Mass spectrometry-based methods to detect toxins
Mass spectrometry (MS) has been used as a method to dissect components of botulinum toxin complexes [37–39]. The MS-based method, called ENDOPEP-MS, uses antibodies to concentrate and extract BoNT from test samples [38]. The concentrated toxins are then subjected to an endopeptidase activity–based assay to generate target cleavage products. Finally, MS is used to identify these products. This approach has been successful in identifying BoNT serotypes A, B, E, and F in various food and clinical matrices with greater sensitivity than the mouse bioassay.
Morineaux et al. [40] recently described a MS method that employs immunocapture enrichment by antibodies specific for BoNT/A-L chains. The enriched analyte is then analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) on a triple quadrupole mass spectrometer (QqQ) in multiple reaction monitoring (MRM) mode. Peptides from BoNT LC specific to the subtypes BoNT/A1–A3 and BoNT/A5–A8 could be identified. BoNT/A subtypes were correctly identified in culture supernatants, water, and orange juice samples with a LOD of 20–150 mouse lethal doses (LDs), but there was a lower sensitivity in serum samples.
Kalb et al. [41] have described the development of a quantitative enzymatic method for the detection of four BoNT serotypes using matrix-assisted laser desorption/ionization—time of flight (MALDI-TOF) MS. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were carefully examined, including the concentration of the substrate and internal standard, the length of time for the cleavage reaction, and the components present in the reaction solution. Longer incubation time produced more sensitive results but was not capable of determining higher toxin concentrations, whereas a shorter incubation time was less sensitive. To address these limitations, a novel two-step analysis was developed [41]. By combining the results from a two-stage quantification, four or five orders of magnitude in dynamic range are observed for detection of BoNT serotypes A, B, D, and F. To minimize the number of cleavage reactions and analytical samples, the assay can be multiplexed using mixtures of different neurotoxin substrates. Numerous different research groups (including Kalb et al. [42], Björnstad et al. [43], and Hines et al. [37]) have used MS to dissect the components of the BoNT/G complex, revealing BoNT/G as well as other toxin protein components, namely NTNH, HA-17, HA-33, and HA-70. Overall, the use of MS can provide rapid and definitive results.
8. Enzymatic assays to detect toxins
Rapidly distinguishing between the presence of active versus inactive toxin is critical for effective medical intervention in toxicoses. As BoNTs are zinc metalloproteases, knowledge of the human targets for these enzymes has enabled development of enzyme-substrate assays. Activity assays have been developed using a wide variety of detection systems. Toxin samples can be treated with recombinant versions of host–target substrates (such as SNAP-25), and the cleavage products can be detected using immunoblotting. Alternatively, fluorogenic peptide substrates emit a signal when cleaved. One such system uses a peptide (“SNAPtide”) with reverse-phase HPLC and a fluorescence detector to detect as low as 5 pg/mL of BoNT/A in skim milk [45]. Other peptide substrates (VAMPtide and SYNTAXtide) have been used for detection of their cognate BoNTs [46]. The levels of substrate cleavage correlate well with toxin activity.
9. Cell-based assays
Cell-based assays measure BoNT receptor binding, translocation, and enzymatic activity and can be
10. New antibody and biosensor technologies
Diamant et al. [55] have used an interesting approach for generating antibodies that have higher specificity against serotypes A, B, and E, and possess neutralizing capabilities. Mice were immunized with a “trivalent mixture” of recombinant fragments of neurotoxins A, B, and E. The method generated numerous different monoclonal antibodies against each serotype. Most of the monoclonal antibodies had higher ELISA titers compared to polyclonal antibodies and had specificities with five orders of magnitude greater specificity. These antibodies also protected against neurotoxin dosages of 10–50 LD50. They also observed a neutralizing synergy when serotype-specific monoclonal antibodies were combined into an oligoclonal mixture.
Detection methods can also utilize highly sensitive antibodies to enrich or enhance sample preparation as well as amplify the signal. For example, an assay with a large immunosorbent surface area (ALISSA) [56, 57] utilizes an antibody to concentrate the neurotoxin onto the surface of a large bead. The “captured” toxin molecules are then used in an enzyme assay. Using food matrices, the LOD for ALISSA was observed as low as 50 fg/mL. This is far more sensitive than the mouse bioassay, immunoassay, or enzyme assay and suggests that it may be useful for detecting food contamination. Marconi et al. [58, 59] have also described the use of surface plasmon resonance (SPR) to examine synaptic vesicle capture by antibodies against BoNT substrates, such as SNAP25 and VAMP2. SPR could be used with cultured neurons in 96-well plates incubated with either BoNT/A or BoNT/B and may be an alternative to animal studies. Further development of label-free and optical biosensors for detecting botulinum toxin [61, 62] will provide additional technologies with possible impact on food safety.
11. Challenges for botulinum neurotoxin detection: new serotypes in the environment
Kull et al. [62] described the isolation of a novel
12. Botulinum neurotoxin detection in the environment: role of climate change and algal blooms in avian botulism and the challenges of environmental matrices
Increased global temperature has been associated with increased algal blooms. The role of these algal blooms in disease is unclear. However, recently, a connection between algal blooms and botulism has been explored. Avian botulism is a disease that often occurs on a yearly cycle and results from the ingestion of neurotoxins by birds. This disease has become increasingly common in the U.S. Great Lakes [64], as have blooms of the green alga
In a follow-up study using PCR, Sadowsky et al. [66] reported that algae mats from different shores of the Great Lakes contained the serotype E gene. Also,
Vidal et al. [67] examined numerous environmental factors that influence the prevalence of the unusual mosaic BoNT serotype C/D. Between 1978 and 2008, 13 avian botulism outbreaks were observed, killing 20,000 birds. A significant association was found between the number of dead birds recorded in each botulism outbreak and the mean temperature in July (with average temperatures being higher than 26°C). The presence of
The presence of
Probably, one of the greatest challenges is determining which environmental matrices should be collected and analyzed, and which ones would provide the most definitive information about potential threats to humans and animals. For instance, Anza et al. [70] examined the role of eutrophication and avian botulism outbreaks in wetlands receiving effluents from urban wastewater treatment plants. Numerous different avian pathogens, including clostridial pathogens, were present in wastewater and could pose a threat to birds living in wastewater wetlands. Methods to detect BoNTs in environmental matrices could be adapted from previous studies of food and clinical samples or may require new technologies. Future studies in this area are clearly warranted.
13. Future technologies to detect botulinum neurotoxins
The discussion herein has presented a general overview of methods currently being used to detect BoNTs. Many current methods to detect BoNTs in food and environmental matrices have been adapted from the clinical laboratory. New possibilities to consider, to name a few, could exploit the tools of nanotechnology, mHealth, and the use of mobile devices, the capability of miniaturization for even more sensitive and rapid detection of BoNTs. The application and practical use of these technologies might be valuable advancements to current methods to detect BoNTs.
14. Conclusions and recommendations
To maintain a safe food supply and to detect toxins in an ever-changing environment, an ongoing, concerted effort in assay development and validation is essential for human health and safety. Some areas for investigators to consider include the development of new antibodies and binding molecules specific to BoNT serotype F as well as new hybrid serotypes. The impact of different types of neurotoxin accessory proteins on the detection of BoNTs should also be examined. Furthermore, the impact of food processing conditions on the stability and bioavailability BoNTs is an area in need of further study. The development of new bioassays based on non-mammalian systems and cell cultures should also be supported as well as the advancement of new portable and field-deployable testing methods, including those based on miniaturization of current bench top instruments. These are only a few recommendations, but their development and use should help to further ensure food safety and animal and human health.
Acknowledgments
This work was funded by the U.S. Department of Agriculture, Agricultural Research Service (National Program 108, Project No. 5325-42000-048-00D). Larry H. Stanker also received funding from the U.S. Department of Homeland Security (Interagency Agreement No. 40768). Kirkwood M. Land was supported by the Department of Biological Sciences at the University of the Pacific.
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