**Technologies for Detecting Botulinum Neurotoxins in Biological and Environmental Matrices**

Luisa W. Cheng, Kirkwood M. Land, Christina Tam, David L. Brandon and Larry H. Stanker

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63064

#### **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), pro‐ duced 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. Here‐ in, 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 num‐ ber of cases of botulism in wildlife. Finally, we consider how environmental changes im‐ pact 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 *Clostridium botulinum*. This pathogen, which is a gram-positive, anaerobic spore-

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forming bacterium, produces botulinum neurotoxins (BoNTs). Humans are susceptible to the effects of these poisons, which are among the most toxic molecules known [1]. The parenteral lethal dosage for humans is 0.1–1 ng/kg, and the oral dose is 1 μg/kg. A single gram of BoNT released into the environment and subsequently inhaled could kill more than one million people [2, 3]. BoNTs exert their biological effects by blocking acetylcholine release by neurons. To date, BoNTs have been divided into seven serotypes, denoted as A through G, of which A, B, E, and F are known to be toxic to humans [4–6]. However, all of the botulinum serotypes are possibly toxic to people. In addition to the principal serotypes, at least 40 additional subtypes have been described based on differences in both primary peptide sequence and three-dimensional structure [4–6]. In this discussion, we review the basic biology of BoNTs and current methods to detect these molecules in biological and environmental matrices.

*C. botulinum* isolates are categorized into different groups [5, 6]. Members of Group I are referred to as "proteolytic" and produce toxin types A, B, or F. They are widely distributed in the environment and often found in various raw foods. BoNTs can cause symptoms at levels as low as 5 ng. Although the onset of symptoms typically takes 12–36 hours, the time course depends on the amount of toxin ingested [2, 3]. It can take much longer for symptoms to manifest. Initial symptoms include diarrhea and vomiting followed by neurological effects that include blurred vision, weakness, and difficulty in swallowing, talking, and breathing. Unless diagnosed early, mortality rates can be as high as 40% [1–4]. Timely response and current treatments have reduced mortality to less than 10%. The most common foods involved in outbreaks are improperly preserved meat or fish products, but a range of other foods have been implicated, such as cheeses (including vegetables preserved in oil and cheese). Because botulinum toxins are not heat stable, they can be inactivated at cooking temperatures.

Strains in Group II are classified as "non-proteolytic" [5, 6]. These *C. botulinum* strains synthesize neurotoxin B, E, or F. These bacteria can grow at temperatures <3°C are ubiquitous in the environment. Moreover, one can find Type E strains in aquatic habitats [1–4]. It is not known whether these strains can synthesize neurotoxins in refrigerated processed foods without visible spoilage. The endospores of strains in this group are not as resistant to heat as those strains in Group I. Neurotoxins synthesized by strains in Group II toxins have shown to be less potent than those of Group I; at least 0.1 μg of neurotoxin is required to cause symptoms of botulism. However, their other biological properties are similar. Foods involved in out‐ breaks of Group II botulism include cold-smoked fish and other preserved fish products.

Group III botulinum produces toxins of serotype C or D and is associated with avian and nonhuman mammalian botulism [5, 6]. Whole genome sequencing analysis indicates that strains of physiological group III are probably more related to *Clostridium haemolyticum* and *Clostri‐ dium novyi* than to *C. botulinum* serotypes belonging to Groups I and II. Group IV is rare and has not been well characterized. However, it does synthesize neurotoxin serotype G.

Bacteriophages contain the neurotoxin genes of *C. botulinum* serotypes C and D [5, 6]. The BoNT prophage replicates in the bacterium as a large plasmid, and strains containing the phage can become toxigenic via either type C or type D phages. The distinction between types C and D is not clear because chimeric sequences have been isolated from the environment. These toxin genes have been identified in avian isolates. They contain sections from both BoNT/C and BoNT/D genes and are referred to as type C/D [5, 6]. The chimeric toxin is more pathogenic to avians than either serotype C or D individually. In *C. botulinum* serotypes C and D, there is a small amount of a binary toxin, denoted as the C2 toxin. The genes encoding the C2 toxin have been localized to a plasmid. Structurally and functionally, the C2 toxin contains a translocation domain and an ADP-ribosylating domain that has been shown to target cellular actin. The occurrence of other chimeric botulinum toxin genes has yet to be determined [5, 6].

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 *C. botulinum*, the neurotoxins are first synthesized as a large holotoxin (approx‐ imately 150 kDa). They are then processed by a trypsin-like protease in *C. botulinum* yielding two polypeptides (one approximately 100 kDa and the other approximately 50 kDa) that are still bound by a single disulfide [2, 3]. The neurotoxin structure mimics other known A–B dimeric toxins found in other bacterial pathogens. The ~100 kDa fragment is called the heavy chain (HC) and aids the binding of the neurotoxin to host cell receptors and its translocation from vesicles to the cytoplasm [2, 3]. The ~50 kDa fragment, called the light chain (LC), contains the enzymatically active domain of the neurotoxin. Recombinantly, expressed LC is routinely used for activity-based neurotoxin assays. Antibodies specific for the HC and LC are used for immunoassays for detecting neurotoxins as well as for neutralization.
