The Florida manatee (Trichechus manatus latirostris) is a threatened marine mammal inhabiting the southeastern United States, primarily the coastal waters of Florida. Lethal boat-strike injuries, loss of warmwater habitat, and brevetoxicosis are the largest threats to the sustainability of the manatee population (Martin et al., 2017; Runge et al., 2017). Harmful algal blooms (HABs) from Karenia brevis in southwest Florida frequently cause mass mortalities of manatees from brevetoxicosis (Landsberg et al., 2009). Extreme low temperatures in the winters of 2009–2010 and 2010–2011 led to record numbers of cold-related manatee deaths and the declaration of unusual mortality events across the state¹ (Hardy et al., 2019). The Marine Mammal Protection Act defines an unusual mortality event (UME) as “a stranding that is unexpected; involves a significant die-off of any marine mammal population; and demands immediate response.”² These previous two UMEs were associated with high manatee mortality primarily in Brevard County, in east-central Florida. Brevard County’s portion of the Indian River Lagoon (IRL) is the most important area for manatee use on the Atlantic Coast (Deutsch et al., 2003), providing essential habitat to manatees year-round, with counts peaking during the spring and seasonal migration. An aerial abundance survey conducted in March 2012 estimated that 70% (95% CI 60–80%) of the manatees on the east coast of Florida were present in Brevard County at the time (Martin et al., 2015).

The IRL has a long history of diverse HABs (Landsberg et al., 2006; Phlips et al., 2015), which include some toxic species that pose a risk to marine mammals (Fire et al., 2015). In 2011, a series of non-toxic bloom events with direct ecosystem disruption resulted in a chain of effects that ultimately triggered a manatee UME in 2013.³ A dominant spring bloom of mixed microalgae in 2011 reduced light penetration, with resultant seagrass losses, poor water quality, and fish and shellfish kills. The following year, brown tide (Aureoumbra lagunensis) in the northern IRL again impaired seagrass growth, resulting in still more losses (Gobler et al., 2013; Phlips et al., 2015; Breininger et al., 2017; Morris et al., 2018). An estimated 47,000 acres (60%) of seagrass was lost during 2009–2012 between Ponce Inlet, in Volusia County, and Fort Pierce Inlet, in St. Lucie County, covering most of the IRL (Lapointe et al., 2015, 2020; Morris et al., 2018). This loss of seagrass caused dietary shifts in manatees to the then predominant available Rhodophyta, ultimately triggering a manatee mass-mortality event.

In July 2012, the first manatees presenting with sudden death of unknown cause had large volumes of Rhodophyta (e.g., Gracilaria spp.) in the gastrointestinal tract. By the end of March 2013, the total number of manatee deaths from natural and undetermined causes (n = 166) had reached a level of more than six times the previous 5-year average (n = 27; excluding the 2009–2010 and 2010–2011 years with high winter mortalities) for this period in Brevard County. Under two of the UME criteria set forth in procedures of section 404 of the Marine Mammal Protection Act, the Working Group on Marine Mammal Unusual Mortality Events determined a UME occurred from 1 January to 16 December 2013.³ The declaration was based on (1) “a marked increase in the magnitude or a marked change in the nature of morbidity, mortality or strandings when compared with prior records” (UME criterion 1) and (2) “affected animals exhibit[ing] similar or unusual pathological findings, behavior patterns, clinical signs, or general physical condition” (UME criterion 5).⁴

Manatees are generalist herbivores that feed on an extensive variety of aquatic vegetation, including macroalgae,^5,6 which are not normally considered a significant portion of the manatee’s diet (Hartman, 1979). Although macroalgae concentrate and produce a wide range of bioactive compounds with toxic and cytostatic, antiviral, antihelmintic, antifungal, or antibacterial activity (Higa and Kuniyoshi, 2000; de Almeida et al., 2011; Kumar and Sharma, 2021), no negative effects associated with the ingestion of macroalgae have been documented in manatees. Furthermore, other than brevetoxicosis and cold stress syndrome, disease is relatively uncommon in wild manatees (Owen et al., 2018).

The investigation of the 2013 manatee UME described here involved a multidisciplinary approach with the objective of determining the cause of mortality and examining any causal factors that contributed to the broad scale of this event. The dietary change associated with the seagrass die-off in the IRL likely resulted in a novel bacterial pathogenesis and a mass-mortality event previously undescribed in a marine mammal species.

Manatees in the IRL are at risk from diverse environmental and anthropogenic factors, with documented mass mortalities from algal biotoxins and cold temperatures and deaths associated with watercraft strikes (Stith et al., 2012; Fire et al., 2015; Hardy et al., 2019). As demonstrated here for the first time, the consequential shift in dietary items directly linked to algal blooms, and a seagrass die-off with resultant change in available forage items, posed a new risk to manatees from clostridial toxins. Associated scenarios with this shift in dominance from seagrass to macroalgae could have been a switch to the ingestion by manatees of toxic, bioactive, or contaminant compounds directly from the macroalgae or of epiphytic toxic microalgae (e.g., dinoflagellates) or associated bacterial pathogens. For example, the macroalga Cladophora in the Great Lakes is a significant reservoir for Shiga toxin–producing Escherichia coli, enterococci, and Clostridium botulinum, with the latter linked to outbreaks of avian botulism in Lake Michigan (Whitman et al., 2003; Ishii et al., 2006; Chun et al., 2013, 2015). Natural antimicrobial compounds produced by macroalgae (Ballantine et al., 1987) could also hypothetically cause a shift in the microflora of the manatee digestive tract leading to impaired health or triggering a dysbiosis.

Initial environmental surveys for potentially toxic microalgae or macroalgae in the IRL did not reveal any toxic candidates that could have fit the case definition. Harmful and toxic microalgae in the IRL include the saxitoxin-producing dinoflagellate Pyrodinium bahamense, occasional incursions by Karenia brevis (which produces brevetoxins), and low-toxicity to non-toxic Pseudo-nitszchia spp. (which produce domoic acid) (Landsberg et al., 2006; Phlips et al., 2015), yet there was no evidence that manatees had been exposed to these neurotoxins during the UME. Benthic dinoflagellates Prorocentrum spp., which produce okadaic acid and dinophysistoxins, utilize macroalgae and seagrass as substrate in the IRL (Anderson, 2002), and although these toxins can cause diarrheic shellfish poisoning (DSP) in humans (Murata et al., 1982), manatee gastrointestinal contents were negative for these toxins. Other than diarrhea, the acute toxemia presentation and the gross and microscopic pathological findings in manatees did not parallel the symptoms associated in humans with DSP.

Macroalgae produce a diverse and complex array of secondary metabolites that deter feeding by marine herbivores (Paul and Puglisi, 2004). A shift of manatees to a predominantly macroalgal diet could have resulted in the inadvertent ingestion of toxic or pathogenic compounds. There is a diverse macroalgal population in the IRL, with Gracilaria tikvahiae, Hypnea musciformis, Acanthophora spicifera, Chaetomorpha sp., Ulva lactuca, and Codium decorticatum the major components of the drift algae that sometimes form high-biomass blooms under eutrophic conditions (Virnstein and Carbonara, 1985; Littler et al., 2008; Whitehouse and Lapointe, 2015; Barile, 2018). Qualitative gut-content analysis of manatee carcasses examined during the UME revealed diverse macroalgae and seagrasses, paralleling the known availability of these forage items in the IRL following the major seagrass die-off of Thalassia testudinum, Halodule wrightii, and Syringodium filiforme triggered by superbloom events in 2012 (Morris et al., 2018). During the manatee UME years and following, dominant macroalgal drift communities in the IRL included Rhodophyta G. tikvahiae, Hypnea musciformis, Hydropuntia secunda and the chlorophyte Chaetomorpha linum, as well as cyanobacterial mats (Lapointe et al., 2015, 2020). Such representative macroalgal taxa were found in manatee gut contents.

Because the human consumption of uncooked Rhodophyta Polycavernosa tsudai (= Gracilaria edulis), G. verrucosa, G. chorda, and G. coronopifolia (Gracilariales) has been associated with fatal food-poisoning incidents in the Pacific region (Halstead and Haddock, 1992; Hanne et al., 1995; Yotsu-Yamashita et al., 2004; Kumar and Sharma, 2021), we focused on testing G. tikhaviae, manatee stomach contents, and feces for polycavernoside. Polycavernosides A and B are the primary toxins associated with human intoxications (Yotsu-Yamashita et al., 1993), and, although intoxications were originally postulated to have originated from macroalgae, those polycavernosides might have been produced by cyanobacteria associated with the macroalgae (Navarro et al., 2015). Macroalgae (and any unidentified epiphytes) and manatee stomach contents and feces tested here were negative for polycavernoside A and were not further considered as possible causal factors. Also, the pathological finding of enterocolitis in manatees was different from that resulting from acute toxicity associated with Gracilaria.

Other compounds implicated in Gracilaria-associated poisoning incidents include prostaglandins, aplysiatoxins, and debromoaplysiatoxins (Fusetani and Hashimoto, 1984; Nagai et al., 1996), but these were not further investigated in the present study. Rhodophytes, predominantly species in the Gigartinales (e.g., Hypnea musciformis), also produce the compound carrageenan, which is widely used as a food additive (Tobacman, 2001; Ciancia et al., 2020). Laboratory rodents and lagomorphs fed rhodophytes Chondrus crispus and Eucheuma spinosum over a 1-month to 1-year period initially developed hemorrhagic and ulcerative lesions, cellular infiltrates, and crypt abscesses. Following chronic exposure, there was a loss of mucosal folds and scarring of the colon, leading to the formation of construction rings. Carrageenan was determined to be the primary compound initiating these lesions (Marcus and Watt, 1969). Carrageenan may also alter the intestinal microflora, potentially leading to a state of dysbiosis (see below). Bacteriological studies of carrageenan-exposed guinea pigs with induced cecal ulcerations showed shifts in the microflora with an increase in coliforms and Gram-negative anaerobes (Onderdonk and Bartlett, 1979), as well as a reduction in anti-inflammatory bacteria together with the enrichment of members of the Clostridiales vadin BB60 group, among other Firmicutes and Tenericutes (Shang et al., 2017). Ingestion by manatees of Rhodophyta possibly containing carrageenan could have predisposed them to ulcerative colitis and contributed to a consequential shift in the intestinal microflora. Further study would be required to quantify the carrageenan, prostaglandin, aplysiatoxin, and debromoaplysiatoxin content of the different macroalgae ingested by manatees and to determine whether these compounds could affect gastrointestinal health.

Our investigation did not include a nutritional analysis of macroalgae collected during environmental surveys, but, in general, seaweeds have been reported to be a valuable food source with high levels of carbohydrates as well as minerals, vitamins, and trace elements (MacArtain et al., 2007). To examine any electrolyte imbalances or deficiencies, vitreous humor was analyzed as conceptualized by Varela and Bossart (2005). Findings in UME manatees included lower Glc and TP and higher UN, Na, and Cl. With due consideration of the limitations when interpreting different analytical methodologies, and given the unknown time of death in the previous study, and that data from only two manatees were available in the present study, these findings excluded acute death from hypocalcemia but considerations included septicemia or endotoxemia, reduced production or loss of serum proteins (e.g., protein-losing enteropathy), or dehydration, all of which support the pathological findings in UME manatees. Furthermore, concentrations of trace minerals in manatee livers were within expected range.

Brevard County is a central hub for manatee migration, largely driven by seasonal changes in temperature (Deutsch et al., 2003). At the peak of the UME in March 2013, a distributional aerial survey documented at least 1,719 manatees in the county (Reynolds and Scolardi, 2016). It is likely that most of these manatees were consuming the same, predominantly macroalgal diet as found in carcasses documented during the UME, yet many survived. Nutritional analyses of manatee diets indicate that macroalgae are generally lower in fiber content than are seagrasses (Siegal-Willott et al., 2010; Rodrigues et al., 2021). Gastrointestinal disorders that have been associated with a low-fiber diet in herbivores include gastric acidosis, dysbiosis, diarrhea, poor digestion, bloat, and displacement of the intestines (Dougal et al., 2014).⁸ An abrupt transition to a macroalgal diet with low fiber content may have made new manatee arrivals in the IRL specifically vulnerable to gastrointestinal dysbiosis, which is supported by the peak in UME carcass numbers during migration in early 2013, the first winter in which manatees encountered predominant Rhodophyta macroalgae. Other hindgut fermenters, such as the horse, can rapidly reverse disruptions in microbial homeostasis following a diet change (Fernandes et al., 2014), and it is plausible that this adaptation eventually occurred in manatees that foraged on macroalgae. Similarly, rumen microbiota of macroalga-consuming North Ronaldsay sheep adapted to efficiently digest this vegetation (Williams et al., 2012). The likely restoration of the gastrointestinal microbial balance after manatees had transitioned to a macroalgal diet is further supported by the decrease in morbidity and mortality cases in the months and years after the peak of the UME.

A multifactorial etiology of a predominant macroalgal diet low in fiber plus the aforementioned macroalgal compounds could have initiated some of the gastrointestinal lesions seen in manatees, with the proximal cause of death being clostridial infection. Macroalgae also produce antimicrobial compounds (de Almeida et al., 2011), so it is possible that the normal intestinal microbiota of manatees was affected by a high macroalgal intake, with an associated increase in Clostridiales. In domestic or captive animal environments and in humans, the initiation of dysbiosis leading to domination by anaerobic clostridia, particularly by Clostridioides difficile, has been linked to antibiotic treatments, hospitalization, and neonatal microbiomes (Antharam et al., 2013; Cassir et al., 2015; Popoff, 2018). In an analogous situation in the wild, dietary shifts or a reduction in forage diversity can contribute to a change in gut microflora, setting up the potential for a dysbiosis and pathobiome (Stecher et al., 2013; Bass et al., 2019). In the horse, a hindgut fermenter similar to the manatee, an abrupt transition from a grain-based diet to pasture grazing caused an immediate change in the relative abundances of the fecal bacteria, including the Clostridiales (Fernandes et al., 2014).

Clostridial gastrointestinal diseases of animals have been well documented, but primarily in domestic livestock, pets, and captive terrestrial mammals (Neiffer, 2001; Bojesen et al., 2006; Rodriguez et al., 2016; Uzal et al., 2016; Capewell et al., 2020; Nyaoke et al., 2020), with few reported cases in terrestrial wildlife (Asaoka et al., 2004; Boujon et al., 2005; Silva et al., 2014), and in captive marine birds (Hines and Dickerson, 1993; Lueders et al., 2017) and marine mammals (Buck et al., 1987; Anderson et al., 2015). Clostridia (e.g., Clostridium perfringens, Clostridioides difficile) are naturally present in the normal gut microbiome and fecal samples of asymptomatic marine mammals, fish, mollusks, and crustaceans (McBee, 1960; Aschfalk and Müller, 2001; Miller et al., 2010; Morris et al., 2011), with ubiquitous distribution in freshwater and marine environments in water, sediment, sewage, and biota and on biological substrates (Miller et al., 2006; Pasquale et al., 2011; Rodriguez et al., 2016). Few cases of lethal clostridial infections have been associated with marine animals, with incidental findings of Clostridium perfringens and P. sordellii suspected in a die-off of the long-spined sea urchin Diadema antillarum (Bauer and Agerter, 1987). Reports of clostridial diseases in free-ranging wild marine animals are less common, with none thus far documented to be the primary cause of mass mortalities in marine mammals. One live-stranded long-beaked common dolphin, Delphinus capensis, beached in La Jolla, California, with systemic infection of C. perfringens type A (Danil et al., 2014), and one stranded sperm whale, Physeter macrocephalus, in Denmark had suspected infection associated with C. septicum (Hansen et al., 2016).

The presence of Clostridia in the hindgut of manatees in the IRL is not unexpected, but no comparative baseline data are available with which to identify shifts to more pathogenic strains or species if manatees are undergoing dysbiosis. Clostridium has been documented to be a dominant bacterial genus in manatees examined from the Crystal River (on Florida’s west coast) and eastern Florida Atlantic (Mao et al., 2011, cited in Merson et al., 2014), but in-depth analysis of clostridial diversity and speciation has not been done. Clostridium butyricum was noted to be one of the 10 most abundant bacterial operational taxonomic units in fecal samples from Crystal River manatees (Merson et al., 2014). In the closely related Antillean manatee, Trichechus manatus manatus, held in captivity in Japan, even under a modified terrestrial herbivorous diet compared to natural conditions, Clostridium spp. were among the top 20 dominant bacteria in fecal samples (Suzuki et al., 2019). Because hindgut fermenters such as manatees have a long intestine, anaerobic conditions suited for Clostridium colonization exist, as most oxygen consumption by microbes takes place in the foregut (Suzuki et al., 2019).

The positive IHC test results for Clostridioides difficile toxin A seen in 13 animals, but the consistent lack of C. difficile PCR detection or isolation, despite repeated efforts using specifically designed primers and specialized agar, may indicate that C. difficile was absent or that the IHC was cross-reacting with P. sordellii hemorrhagic toxin. The chemical similarity between C. difficile toxin A and P. sordellii hemorrhagic toxin has been identified and immunological cross-reactivity recognized, with both species 71% homologous at the genomic level (Martinez and Wilkins, 1988; Scaria et al., 2015). We have not found any cases reporting on this difficulty in interpreting possible false positives of C. difficile IHC toxin A in P. sordellii. But two ELISA tests conducted within 24 h of death confirmed C. difficile toxins A/B, and the clinical presentation, gross and microscopic findings, and the evident positive IHC tests for C. difficile toxin A in 13 manatees (if not cross reacting) is reminiscent of C. difficile–associated disease (CDAD) in horses and pigs (Songer, 2004; Songer and Uzal, 2005; Songer et al., 2009), possibly implicating C. difficile in the pathogenesis of the clostridial infections of manatees. Inflammation, necrosis, and intercryptal exudates of fibrin and neutrophils associated with CDAD (Keel and Songer, 2006; Uzal and Songer, 2019) and P. sordellii described in pigs and horses (Diab et al., 2013; Nyaoke et al., 2020, respectively) were commonly observed microscopically at varying degrees of severity in the present study. Mucosal and submucosal edema, similar to CDAD in horses, was also observed in this study and associated with C. difficile toxin A by IHC in 13 manatees.

Virulence factors determined in the P. sordellii strain FL-manatee genome were diverse, but large clostridial toxin genes (lethal and hemorrhagic) were not detected. In combination with the pathogenic potential of the other clostridial species (with their toxins or toxin genes) detected in symptomatic manatees, this suite of factors could have contributed to the overall clinical presentation of lethal clostridial infection. Numerous Clostridium perfringens toxins including kappa toxin and theta toxins have been implicated in clostridial pathogenicity (Rood, 1998). Perfringolysin O (PFO, theta toxin) in C. perfringens, a cholesterol-dependent cytolysin, synergistically acts with C. perfringens alpha toxin to trigger platelet and leucocyte aggregation in cases of gas gangrene, resulting in the formation of blood vessel obstruction, hypoxia, and tissue destruction (Verherstraeten et al., 2013). It contributes indirectly to toxic shock by releasing inflammatory cytokines, and is involved in bovine enterotoxemia and necrohemorrhagic enteritis (Verherstraeten et al., 2013, 2015; Popoff, 2014). PFO was predicted to be encoded by the ORF_02133 gene in P. sordellii strain FL-manatee. We also sequenced kappa toxin (or collagenase) encoding genes within the P. sordellii strain FL-manatee genome (ORF_01190). Although the role of collagenase in virulence is not known, host collagen remodeling by clostridial bacteria is considered to enhance tissue colonization (Eckhard et al., 2011). An experiment showed that 0.7 μg of purified C. perfringens collagenase produced a definite hemorrhage on rabbit skin, while 30 μg killed mice from associated severe lung hemorrhage (Kameyama and Akama, 1971). The role of hemolysins A and C (ORF_00534, ORF_01294, and ORF_02946) in the P. sordellii isolate from manatees is unknown, but in other bacteria, these are major virulence factors. For example, these hemolysins contribute to mucosal damage in the colon of pigs with swine dysentery (Bellgard et al., 2009), and together with other hemolysins, they have been identified in C. butyricum causing neonatal necrotizing enterocolitis (Dong et al., 2020). In P. sordellii strain FL-manatee, the gene ORF_03500 was predicted to encode phospholipase C (CpPLC), which is homologous to C. perfringens alpha toxin but displays lower enzymatic and hemolytic activity (Vidor et al., 2015). But the role of C. perfringens alpha toxin in enteric disease of mammals and birds is still unknown. Although that toxin might have been involved in the enteric lesions of the manatees studied in the present study, whether it did remains speculative.

The P. sordellii strain FL-manatee gene ORF_01874 was predicted to encode sialidases (neuraminidases), which could play a role in the notable heterophilic and lymphoplasmacytic inflammatory response in manatees. Paeniclostridium sordellii infections are characterized by the profound capillary-leak syndrome, development of a leukemoid reaction (LR), and a marked shift to a granulocytic population; in humans, the LR is a hallmark and predictor of a fatal outcome (Aldape et al., 2007). A leukemoid reaction is also a characteristic of Clostridioides difficile infections (Marinella et al., 2004). Clostridium perfringens strains also typically possess three sialidase-encoding genes. In vitro, the sialidases can enhance binding and cytotoxic effects of epsilon toxin in type C. perfringens type D isolates, contributing to intestinal colonization and increasing toxin action (Li et al., 2011). In vivo, NanI (the main sialidase produced by C. perfringens) increases colonization of C. perfringens type F and the effect of its enterotoxin (CPE) (Li et al., 2021). Sialidase production has also been implicated in the virulence of other pathogenic bacteria, such as Vibrio cholerae (enhancing the activity of cholera toxin by increasing toxin binding levels; Galen et al., 1992) and Streptococcus pneumoniae (assisting in growth, biofilm formation, and exposing adhesion sites; King, 2010).

Adhesion and colonization of animal tissue by bacteria is an important step in establishing infection. Intestinal colonization is a complicated process that involves several colonization factors. The gene that encodes fibronectin-binding protein A (FbpA and Fbp68) was identified in the P. sordellii strain FL-manatee (ORF_03002). FbpA is described as an adhesive protein with a minor but not insignificant role in colonization, implicated in the first step of the intestinal infection by Clostridioides difficile (Barketi-Klai et al., 2011). This role is shared with multiple other adhesins, such as collagen-binding protein (Cna). Cna promotes bacterial adhesion to collagen-rich tissue and therefore contributes to tissue tropism and may influence host species preference due to differences in species-specific collagen-binding affinities (Jost et al., 2006). The Cna protein was implicated as a potential virulence factor in porcine enteritis caused by Clostridium perfringens (Jost et al., 2006) and was correlated with the ability of a C. perfringens strain to cause necrotic enteritis in chickens (Wade et al., 2015). The P. sordellii strain FL-manatee gene ORF_02554 was predicted to encode a Cna protein. Heat-shock GroEL proteins are cell-surface proteins also involved in clostridial cell adhesion and were predicted to be encoded by the P. sordellii strain FL-manatee gene ORF_03377. GroEL has adhesive function in Clostridioides difficile (Hennequin et al., 2001).

Paeniclostridium sordellii, with the two large clostridial toxins, lethal and hemorrhagic, is usually associated with gas gangrene (myonecrosis), sepsis, or fatal toxic shock syndrome in humans (Popoff, 2018), with fewer cases restricted to intestinal disease (de la Fe et al., 2006). In domestic animals, the role of P. sordellii in enteric disease is not fully known, although it has been suggested that it is associated with enterocolitis in horses (Nyaoke et al., 2020). Overall, Clostridia were not detected in manatees in non-digestive-tract lesions, with evidence for three known clostridial pathogens (P. sordellii, C. difficile, and Clostridium perfringens type A), likely working in synergy or by as yet undetermined etiological mechanisms to cause lethal enterocolitis in manatees. A pathogenic role of P. sordellii is proposed based on several lines of evidence, but further research is required. Despite analyses of 17 manatees, we were unable to detect large clostridial toxins in P. sordellii strain FL-manatee but did detect the lethal gene directly from the small intestine of one manatee. Challenges in the detection of lethal (TscL) or hemorrhagic (TscH) toxins in P. sordellii, or evident absence of such toxins, have been described in human cases (Couchman et al., 2015), in hemorrhagic and necrotizing enteropathy in dogs (Capewell et al., 2020), and enterocolitis in horses (Nyaoke et al., 2020), indicating that other virulence factors are involved or that knowledge of virulence factors is incomplete or that diagnostic assays need to be improved for detection. Most strains of P. sordellii do not produce TscL or TscH toxins but are pathogenic (Vidor et al., 2015) and have been associated with necrotizing gastroenteric disease, toxicosis, and mortality in horses, cattle, sheep, dogs, lions, and chickens (Lewis and Naylor, 1998; de la Fe et al., 2006; Rimoldi et al., 2015; Capewell et al., 2020).

New reports and reappraisals of knowledge of the role of Clostridia in animal disease are recognizing that perhaps some virulence factors have been underrecognized for their pathogenesis or that other factors may be at play when known lethal toxins appear to be absent (Goossens et al., 2017). Toxins on plasmids may have been missed, or other factors are significant.

There are few reports of clostridial disease in manatees. A 1-month-old Amazonian manatee, Trichechus inunguis held in captivity, presented with necrohemorrhagic colitis and pneumatosis intestinalis associated with a C. perfringens type A infection (Neto et al., 2016). In managed care of manatees, gastrointestinal disorders from enterocolitis are among the most severe conditions and, undiagnosed, can lead to sepsis, shock, and death⁹ (Neto et al., 2016). Although C. perfringens type A was detected in 11 cases in the present study, no obvious gross or microscopic lesions with gas bubbles were seen, and the blebbing seen in the cecum of three manatees and colon of 11 manatees, reflecting edema-associated blistering and mucosal lifting similar to CDAD associated with Clostridioides difficile (Songer et al., 2009), was striking. In contrast with previous reports that clostridial infection mostly affected manatee calves, this UME included an unusually high proportion of adults. An investigation into P. sordellii–associated sudden death in flocks of sheep found the most severe lesions in young lambs, but the adults affected had all undergone a change in feeding management (Lewis and Naylor, 1998). Adult manatees may be more sensitive to dietary changes and subsequent dysbiosis due to their increased nutritional demand and larger volume of vegetation ingested. In addition, infants could be refractory to CDAD because they lack toxin receptors (Borriello and Wilcox, 1998; Keel and Songer, 2006).

The suite of clostridial virulence factors and toxins identified here, likely caused by a shift to a predominantly macroalgal diet, appears to play a significant role in the mortality of manatees, primarily in the IRL, but also elsewhere. Concern for manatee health and survival warrants further investigation as to primary causative factors, other as-yet-undetermined contributing factors, bacterial source origins, and links to dramatic ecosystem functional changes and stressors. Potential exogenous sources of Clostridiales are unknown, but numerous wastewater-treatment plants, sewage-disposal systems, agricultural activities, and aquaculture operations near the IRL could be of concern.

The IRL is an ecosystem in distress, and, directly or indirectly, the changes in the stability of a primary food source for manatees has had unforeseen consequences that have persisted for the past decade. Further unprecedented declines in seagrass and macroalgae culminated in a manatee UME related to starvation in December 2020,¹⁰ which is ongoing at the time of writing. The findings of Paeniclostridium sordellii, Clostridium perfringens type A, and Clostridioides difficile toxin A in 7, 11, and 13 manatees, respectively, presenting with pathological findings fitting the UME case definition illustrate an important emerging issue for marine animal health and document the expansion of known pathogenesis of Clostridia in marine ecosystems. The risk of clostridial infection to marine mammals is herein highlighted and recognized. Manatees are increasingly at risk from compounding environmental stressors that together pose uncertainties for the long-term stability of this threatened species.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, CP080291 and CP080292; https://www.ncbi.nlm.nih.gov/sra, SRR17734612 and SRR17734613.

Ethical review and approval was not required for the animal study because this research involved diseased animals, not killed for this study, and was conducted by authorized personnel in accordance with section 6 of the Endangered Species Act [implementing regulations 50 CFR 17.21(c) and 50 CFR 17.31(b)] and section 109(h) of the Marine Mammal Protection Act (implementing regulation 50 CFR 18.22).

JL and MW directed the research and investigative component of the UME, and drafted the manuscript. MW conducted and oversaw the manatee necropsies and collected manatee samples. MT prepared and analyzed samples for microbiology and ELISA toxin testing. DR provided histopathological analyses. KS, TR, and TW conducted genome sequencing and annotation. NS performed cytology and biochemistry analyses and coordinated microbiology samples submitted to the University of Florida. PW conducted Clostridium toxin immunohistochemistry. JL evaluated histological and toxin immunohistochemical slides and interpreted results. YK assisted with microbiological analyses and interpretation of toxin immunohistochemical slides. FU conducted and interpreted Clostridium immunohistochemistry and provided ELISA toxin testing. All authors contributed to writing the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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