**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 *Cladophora*, which can serve as a potential habitat for *C. botulinum*. The interactions between *Cladophora* and *C. botulinum* are unclear due to the complex food web associated with this disease. Investigators in several recently published studies [64–66] reported a high number of botulism cases in shoreline birds in Lake Michigan. This increased incidence was correlated with increasingly large accumulations of *Cladophora* in the water. Sadowsky et al. [65] examined algal mats that were collected from Lakes Michigan, Ontario, and Erie in 2011–2012 and then compared them with algal populations in sand and water. They found that 96% of *Cladophora* mats collected from the shorelines in 2012 contained *C. botulinum* Type E. Among the algae samples contain‐ ing detectable *C. botulinum*, the large number of detected *C. botulinum* type E cells indicated that *Cladophora* mats are principal sources of this pathogen. Mouse toxin and antitoxin bioassays further confirmed the toxin in collected samples as serotype E. Further examination of *Cladophora-*associated *C. botulinum* may lead to a model system to study algal–clostridial interactions and result in lower bird mortality.

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, *C. botulinum* was found to be present in amounts of up to 15,000 cells per gram of dried algae, based on quantitation of gene copies encoding serotype E. Moreover, genes for serotypes A and B, which are associated with human diseases, were detected in several of the algal samples. Using mouse toxin assays and subsequent neutralization assays, it was confirmed that *Cladophora*-associated *C. botulinum* was serotype E. One might consider that with increased incidence of extreme drought and other environmental changes, algal blooms may happen more often in water-restricted areas, and *C. botulinum* growth may pose a threat to humans if toxin is produced in algal mats. Developing sensitive detection methods for toxins within algal matrices is urgent, as is monitoring other matrices that could provide an environment for botulinum toxin production. The increased avian botulism associated with increased algal blooms highlights the need to develop new technologies for detection of toxin in the environment, or a re-evaluation of current methods and their use in environmental matrices.

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 *C. botulinum* type C/D in wetland sediments was detected by qPCR. Furthermore, low concentrations of chloride ions and high organic matter content were correlated with the presence of *C. botulinum*. The digestive tracts of dead birds found during botulism outbreaks were also analyzed; *C. botulinum* was present in almost 40% of the studied samples. Recently, Le Maréchal et al. [68] examined livers from dead birds suspected of having botulism and showed that this organ can serve as a reliable matrix for RT-PCR confirmation of disease. This finding may provide wildlife investigators with a faster method to confirm avian deaths due to botulism.

The presence of *C. botulinum* was detected in aquatic invertebrates and flesh-eating inverte‐ brates collected around bird carcasses. Moreover, the presence of *C. botulinum* bacteria in the adult fly stage of some invertebrates raises the question of whether flies can transport *C. botulinum* from one carcass to another. The same investigators examined whether adult blowflies could play a significant role in botulism outbreaks by carrying *C. botulinum* between carcasses. A field experiment and subsequent laboratory tests determined that blowflies could transport *C. botulinum* Type C/D between carcasses [69]. These results confirm that adult flesheating flies could play a role in avian botulism outbreaks. An environmental monitoring protocol for botulinum-carrying flies has not yet been established. It is a matter for future research to determine whether these or other insects could serve as mechanical vectors for botulinum isolates that pose greater threats to humans than the avian isolates.

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.
