Novel Lethal Clostridial Infection in Florida Manatees (Trichechus manatus latirostris): Cause of the 2013 Unusual Mortality Event in the Indian River Lagoon

May combine two claims.

The loss of seagrass in the Indian River Lagoon caused dietary shifts in manatees to the then predominant available Rhodophyta, ultimately triggering a manatee mass-mortality event.

“This loss of seagrass caused dietary shifts in manatees to the then predominant available Rhodophyta, ultimately triggering a manatee massmortality event.”

• cites Lapointe et al., 2015 sourceNot checked — no full text of the source
• cites Morris et al., 2018 sourcePartly supported

  The passage labeled Abstract (page not provided) reports a loss of more than 60% of seagrass coverage in the affected Indian River Lagoon area. It does not report that manatees switched to Rhodophyta or that such a dietary shift triggered a manatee mass-mortality event; searches for those points found no supporting passage. The mortality it mentions concerns finfish and shellfish. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Rhodophytes, predominantly species in the Gigartinales (e.g., Hypnea musciformis), produce carrageenan, which is widely used as a food additive.

“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).”

• cites Tobacman, 2001 sourcePartly supported

  The introduction states that carrageenan is extracted from red seaweed and is widely used as a food additive. The Characteristics of Carrageenan table lists Chondrus, Gigartina, and Eucheuma as predominant sources, but our copy does not identify Gigartinales as the predominant order or name Hypnea musciformis. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Ciancia et al., 2020 sourcePartly supported

  The Carrageenans section says Hypnea musciformis produces large amounts of κ-carrageenan and places its family among the carrageenan-producing Gigartinales. The Concluding Remarks say carrageenans are produced in major quantities only by algae from that order. Our copy does not state that carrageenan is widely used as a food additive. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

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.

“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).”

• cites Dougal et al., 2014 sourcePartly supported

  The Introduction (page not supplied) reports that high-starch feeding in horses alters colonic microbes and lowers pH, supporting the claim’s dysbiosis and acidity components. The Discussion (page not supplied) suggests that diets other than high-fibre diets may increase metabolic-dysfunction risk, but the copy does not associate low fibre with diarrhea, poor digestion, bloat, or intestinal displacement, or establish the full list for herbivores generally. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Rumen microbiota of macroalgae-consuming North Ronaldsay sheep adapted to efficiently digest this vegetation.

“Similarly, rumen microbiota of macroalgaconsuming North Ronaldsay sheep adapted to efficiently digest this vegetation (Williams et al., 2012).”

• cites Williams et al., 2012 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

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, with ubiquitous distribution in freshwater and marine environments in water, sediment, sewage, and biota and on biological substrates.

“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”

• cites McBee, 1960 sourceNot checked — no full text of the source
• cites Aschfalk and Müller, 2001 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Miller et al., 2010 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Clostridial gastrointestinal diseases of animals have been well documented, but primarily in domestic livestock, pets, and captive terrestrial mammals, with few reported cases in terrestrial wildlife, and in captive marine birds and marine mammals.

“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”

• cites Neiffer, 2001 sourceNot checked — no full text of the source
• cites Bojesen et al., 2006 sourceNot checked — no full text of the source
• cites Rodriguez et al., 2016 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

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.

“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).”

• cites Antharam et al., 2013 sourcePartly supported

  The Introduction links antibiotic-induced disruption of the human gut microbiota to C. difficile proliferation, and the Discussion reports that antibiotic exposure during hospitalization was strongly associated with subsequent infection (pages unavailable). The paper does not establish this pattern in domestic or captive animal environments or link it to neonatal microbiomes. It reports depletion of several normally abundant anaerobic bacteria, not general domination by anaerobic clostridia. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Cassir et al., 2015 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Popoff, 2018 sourceNot checked — no full text of the source

May combine two claims.

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.

“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).”

• cites Martinez and Wilkins, 1988 sourceNot checked — no full text of the source
• cites Scaria et al., 2015 sourcePartly supported

  The Background reports immunological cross-reactivity between toxins of C. difficile and C. sordellii. The Results and discussion section on gene repertoire comparison reports homologous toxin proteins, including a C. sordellii hemorrhagic toxin, but does not establish the claimed chemical similarity between the specific toxins or state that the species are 71% homologous at the genomic level. Page numbers are unavailable in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Most strains of P. sordellii do not produce TscL or TscH toxins but are pathogenic and have been associated with necrotizing gastroenteric disease, toxicosis, and mortality in horses, cattle, sheep, dogs, lions, and chickens.

“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).”

• cites Vidor et al., 2015 sourceNot checked — no full text of the source
• cites Lewis and Naylor, 1998 sourcePartly supported

  The Discussion reports C. sordellii as a cause of sudden death in sheep and recognizes it as a cattle pathogen; the Laboratory findings describe necrotic lesions in a sheep’s abomasum. The paper does not establish the cited association across horses, dogs, lions, and chickens. The statement about most strains lacking TscL or TscH is cited to Vidor et al., not to Lewis and Naylor. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites de la Fe et al., 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Other hindgut fermenters, such as the horse, can rapidly reverse disruptions in microbial homeostasis following a diet change.

“Other hindgut fermenters, such as the horse, can rapidly reverse disruptions in microbial homeostasis following a diet change (Fernandes et al., 2014)”

• cites Fernandes et al., 2014 sourcePartly supported

  The Discussion reports that horses moved from a conserved forage-grain diet to pasture developed faecal bacterial communities similar to those of pasture-fed horses within four days. This supports rapid change after a diet switch, but the study does not establish that microbial homeostasis had been disrupted or that a prior state was restored; its dietary challenge was moderate and primarily forage-based. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

One live-stranded long-beaked common dolphin, Delphinus capensis, beached in La Jolla, California, with systemic infection of C. perfringens type A, and one stranded sperm whale, Physeter macrocephalus, in Denmark had suspected infection associated with C. septicum.

“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).”

• cites Danil et al., 2014 sourceSupported
• cites Hansen et al., 2016 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Clostridium butyricum was noted to be one of the 10 most abundant bacterial operational taxonomic units in fecal samples from Crystal River manatees.

“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).”

• cites Merson et al., 2014 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Macroalgae also produce antimicrobial compounds, so it is possible that the normal intestinal microbiota of manatees was affected by a high macroalgal intake, with an associated increase in Clostridiales.

“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.”

• cites de Almeida et al., 2011 sourceSupported

May combine two claims.

Either large numbers of both Clostridium perfringens and Paeniclostridium sordellii were observed on the mucosa of the proximal colon (n = 3), or only P. sordellii was positive in the proximal colon (n = 1).

“Either large numbers of both bacteria were observed on the mucosa of the proximal colon (n = 3), or only P. sordellii was positive in the proximal colon (n = 1).”

Probably not a substantial claim: may not pass S3 (load-bearing).

No organic compounds were identified by mass-spectral-library match of stomach contents (n = 2).

“No organic compounds were identified by mass-spectral-library match of stomach contents (n = 2).”

May combine two claims.

The gastrointestinal compartments of manatee carcasses were filled with diverse macroalga species, and the contents were notably more fluid than usual.

“The gastrointestinal compartments of manatee carcasses were filled with diverse macroalga species, and the contents were notably more fluid than usual.”

May combine two claims.

There was no evidence that manatees had been exposed to saxitoxin, brevetoxins, or domoic acid during the 2013 unusual mortality event.

“yet there was no evidence that manatees had been exposed to these neurotoxins during the UME.”

May combine two claims.

Gram-positive plump rods were present superficially and within the mucosa (n = 7) and in the submucosa (n = 3).

“Gram-positive plump rods were present superficially and within the mucosa (n = 7) and in the submucosa (n = 3).”

May combine two claims.

The lung parenchyma was wet indicating edema, and the airways were filled with fluid, mucus, or froth.

“The lung parenchyma was wet indicating edema, and the airways were filled with fluid, mucus, or froth (Figure 4F).”

May combine two claims.

Clostridioides difficile toxin A was detected in 13 of 16 manatees, in multiple gastrointestinal tissues tested.

“Clostridioides difficile toxin A was detected in 13 of 16 manatees, in multiple gastrointestinal tissues tested: cecum (100%, n = 5, localized staining), mid small intestine (100%, n = 2), distal small intestine (100%, n = 4), distal colon (100%, n = 2), mid colon (100%, n = 1), and proximal colon (73%, n = 11).”

May combine two claims.

The lethal toxin gene of Paeniclostridium sordellii was detected from DNA samples extracted directly from contents of the small intestine (n = 1) but not from those identified as P. sordellii isolates.

“The lethal toxin gene of P. sordellii was detected from DNA samples extracted directly from contents of the small intestine (n = 1) but not from those identified as P. sordellii isolates.”

May combine two claims.

The intestinal content was watery in the small intestine (n = 117), cecum (n = 127) and/or proximal colon (n = 117).

“The intestinal content was watery in the small intestine (n = 117), cecum (n = 127) and/or proximal colon (n = 117) (Figure 4B).”

May combine two claims.

Microscopic lesions were prominent in the small intestine, cecum, and colon, and included one or more of the following changes: necrosis, edema, inflammation, hemorrhage, and mucosa-associated lymphoid tissue depletion or hyperplasia.

“Microscopic lesions were prominent in the small intestine, cecum, and colon, and included one or more of the following changes: necrosis, edema, inflammation, hemorrhage, and mucosa-associated lymphoid tissue depletion or hyperplasia (Table 3 and Figures 5A-C).”

May combine two claims.

Heavy metals in liver samples were negative or within reference range (n = 3, of which one was a control), and tests of trace minerals were unremarkable.

“Heavy metals in liver samples were negative or within reference range (n = 3, of which one was a control), and tests of trace minerals were unremarkable.”

May combine two claims.

Manatee gastrointestinal contents were negative for okadaic acid and dinophysistoxins.

“manatee gastrointestinal contents were negative for these toxins.”

May combine two claims.

Manatee stomach contents were negative for polycavernoside A (n = 10), brevetoxin (n = 5), saxitoxin (n = 15), okadaic acid (n = 5), and domoic acid (n = 5).

“Manatee stomach contents were negative for polycavernoside A (n = 10), brevetoxin (n = 5), saxitoxin (n = 15), okadaic acid (n = 5), and domoic acid (n = 5).”

May combine two claims.

The alpha toxin gene of Clostridium perfringens was detected in the small intestine (n = 7) and in the colon (n = 6).

“Among these, the alpha toxin gene was detected in the small intestine (n = 7) and in the colon (n = 6).”

May combine two claims.

Microbiological, cytological, immunohistochemical, and molecular analyses of Clostridiales from intestinal samples identified Clostridioides difficile toxin A, toxins A/B and toxin A gene.

“Microbiological, cytological, immunohistochemical, and molecular analyses of Clostridiales from intestinal samples identified Clostridioides difficile toxin A, toxins A/B and toxin A gene; Paeniclostridium”

May combine two claims.

Virulence factors determined in the P. sordellii strain FL-manatee genome were diverse, but large clostridial toxin genes (lethal and hemorrhagic) were not detected.

“Virulence factors determined in the P. sordellii strain FLmanatee genome were diverse, but large clostridial toxin genes (lethal and hemorrhagic) were not detected.”

May combine two claims.

Despite analyses of 17 manatees, large clostridial toxins were not detected in P. sordellii strain FL-manatee, but the lethal gene was detected directly from the small intestine of one manatee.

“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.”

May combine two claims.

Mucosal and submucosal edema, similar to C. difficile-associated disease in horses, was also observed in this study and associated with C. difficile toxin A by immunohistochemistry in 13 manatees.

“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.”

May combine two claims.

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.

“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.”

• cites Hennequin et al., 2001 sourceNot checked — no full text of the source

May combine two claims.

Findings in unusual mortality event manatees included lower glucose and total protein and higher urea nitrogen, sodium, and chloride.

“Findings in UME manatees included lower Glc and TP and higher UN, Na, and Cl.”

May combine two claims.

The presence of Clostridia in the hindgut of manatees in the Indian River Lagoon 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.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The P. sordellii strain FL-manatee gene ORF_02554 was predicted to encode a Cna protein.

“The P. sordellii strain FL-manatee gene ORF_02554 was predicted to encode a Cna protein.”

May combine two claims.

The positive immunohistochemistry 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 immunohistochemistry was cross-reacting with P. sordellii hemorrhagic toxin.

“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.”

May combine two claims.

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 C. difficile-associated disease, was striking.

“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.”

• cites Songer et al., 2009 sourceNot checked — no full text of the source

May combine two claims.

The dietary change associated with the seagrass die-off in the Indian River Lagoon likely resulted in a novel bacterial pathogenesis and a mass-mortality event previously undescribed in a marine mammal species.

“The dietary change associated with the seagrass dieoff in the IRL likely resulted in a novel bacterial pathogenesis and a mass-mortality event previously undescribed in a marine mammal species.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Other than brevetoxicosis and cold stress syndrome, disease is relatively uncommon in wild manatees.

“Furthermore, other than brevetoxicosis and cold stress syndrome, disease is relatively uncommon in wild manatees (Owen et al., 2018).”

• cites Owen et al., 2018 sourceNot checked — no full text of the source

May combine two claims.

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.

“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.”

May combine two claims.

The 2013 manatee unusual mortality event followed a dramatic reduction of seagrass coverage in the Indian River Lagoon due to chronic non-toxic phytoplankton blooms, with a resultant ecosystem shift to mixed macroalgal dominance.

“This UME followed a dramatic reduction of seagrass coverage in the IRL due to chronic non-toxic phytoplankton blooms, with a resultant ecosystem shift to mixed macroalgal dominance.”

May combine two claims.

A dominant spring bloom of mixed microalgae in 2011 reduced light penetration, with resultant seagrass losses, poor water quality, and fish and shellfish kills.

“A dominant spring bloom of mixed microalgae in 2011 reduced light penetration, with resultant seagrass losses, poor water quality, and fish and shellfish kills.”

May combine two claims.

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.

“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.”

• cites Marcus and Watt, 1969 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

A case definition was developed to allow standardization of cases in the investigation of the unusual mortality event and associated carcasses between 2012 and 2019.

“A case definition was developed to allow standardization of cases in the investigation of the UME and associated carcasses between 2012 and 2019.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Carrageenan was determined to be the primary compound initiating these lesions.

“Carrageenan was determined to be the primary compound initiating these lesions (Marcus and Watt, 1969).”

• cites Marcus and Watt, 1969 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Polycavernosides might have been produced by cyanobacteria associated with the macroalgae.

“those polycavernosides might have been produced by cyanobacteria associated with the macroalgae (Navarro et al., 2015).”

• cites Navarro et al., 2015 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

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.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

There is a diverse macroalgal population in the Indian River Lagoon, with Gracilaria tikvahiae, Hypnea musciformis, Acanthophora spicifera, Chaetomorpha sp., Ulva lactuca, and Codium decorticatum the major components of drift algae that sometimes form high-biomass blooms under eutrophic conditions.

“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”

• cites Virnstein and Carbonara, 1985 sourceNot checked — no full text of the source
• cites Littler et al., 2008Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Whitehouse and Lapointe, 2015 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Manatee carcasses were examined and processed according to standardized procedures.

“carcasses were examined and processed according to standardized procedures (Bonde et al., 1983).”

• cites Bonde et al., 1983Not checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Carrageenan may alter the intestinal microflora, potentially leading to a state of dysbiosis.

“Carrageenan may also alter the intestinal microflora, potentially leading to a state of dysbiosis (see below).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Polycavernosides A and B are the primary toxins associated with human intoxications.

“Polycavernosides A and B are the primary toxins associated with human intoxications (Yotsu-Yamashita et al., 1993)”

• cites Yotsu-Yamashita et al., 1993 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Negative control slides were prepared in parallel for each paraffin section and treated the same way, except no antibodies were applied.

“Negative control slides were prepared in parallel for each paraffin section and treated the same way, except no antibodies were applied to the slides.”

Classified as probably not a substantial claim. May combine two claims.

Intestinal samples were tied firmly with a string on each end and excised so that intestine contents remained intact, minimizing cross-contamination.

“sections (about 30 cm long) of the small intestine or colon from fresh carcasses were tied firmly with a string on each end, then the tied ends were excised from the gastrointestinal tract so that the intestine contents in the samples remained intact.This measure minimized cross-contamination of intestine contents or fecal material between samples.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Macroalgae produce a diverse and complex array of secondary metabolites that deter feeding by marine herbivores.

“Macroalgae produce a diverse and complex array of secondary metabolites that deter feeding by marine herbivores (Paul and Puglisi, 2004).”

• cites Paul and Puglisi, 2004 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Carcass information was stored in a relational database, double-verified after entry, and reviewed for consistency.

“Carcass information (including sex, length, weight, and cause of death) and recovery data were stored in a relational database, double-verified after entry, and reviewed for consistency.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A shift of manatees to a predominantly macroalgal diet could have resulted in the inadvertent ingestion of toxic or pathogenic compounds.

“A shift of manatees to a predominantly macroalgal diet could have resulted in the inadvertent ingestion of toxic or pathogenic compounds.”

May combine two claims.

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.

“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.”

May combine two claims.

It is likely that most of these manatees were consuming the same, predominantly macroalgal diet as found in carcasses documented during the unusual mortality event, yet many survived.

“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.”

May combine two claims.

The clinical presentation, gross and microscopic findings, and the evident positive immunohistochemistry tests for C. difficile toxin A in 13 manatees (if not cross reacting) is reminiscent of C. difficile-associated disease in horses and pigs, possibly implicating C. difficile in the pathogenesis of the clostridial infections of manatees.

“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. difficileassociated 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.”

• cites Songer, 2004 sourceNot checked — no full text of the source
• cites Songer and Uzal, 2005 sourceNot checked — no full text of the source
• cites Songer et al., 2009 sourceNot checked — no full text of the source

May combine two claims.

Overall, Clostridia were not detected in manatees in nondigestive-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.

“Overall, Clostridia were not detected in manatees in nondigestive-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.”

May combine two claims.

A pathogenic role of P. sordellii is proposed based on several lines of evidence, but further research is required.

“A pathogenic role of P. sordellii is proposed based on several lines of evidence, but further research is required.”

Probably not a substantial claim: may not pass S3 (load-bearing).

At the peak of the unusual mortality event in March 2013, a distributional aerial survey documented at least 1,719 manatees in Brevard County.

“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).”

• cites Reynolds and Scolardi, 2016Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Clostridium has been documented to be a dominant bacterial genus in manatees examined from the Crystal River and eastern Florida Atlantic, but in-depth analysis of clostridial diversity and speciation has not been done.

“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.”

• cites Mao et al., 2011, cited in Merson et al., 2014Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

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.

“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).”

• cites Suzuki et al., 2019 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

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.

“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).”

• cites Neto et al., 2016 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

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.

“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).”

• cites Stecher et al., 2013 sourceNot checked — no full text of the source
• cites Bass et al., 2019 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

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.

“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).”

• cites Suzuki et al., 2019 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

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.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Brevard County is a central hub for manatee migration, largely driven by seasonal changes in temperature.

“Brevard County is a central hub for manatee migration, largely driven by seasonal changes in temperature (Deutsch et al., 2003).”

• cites Deutsch et al., 2003Not checked — no full text of the source

May combine two claims.

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.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

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 longspined sea urchin Diadema antillarum.

“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 longspined sea urchin Diadema antillarum (Bauer and Agerter, 1987).”

• cites Bauer and Agerter, 1987Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

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 unusual mortality event case definition illustrate an important emerging issue for marine animal health and document the expansion of known pathogenesis of Clostridia in marine ecosystems.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Adult manatees may be more sensitive to dietary changes and subsequent dysbiosis due to their increased nutritional demand and larger volume of vegetation ingested.

“Adult manatees may be more sensitive to dietary changes and subsequent dysbiosis due to their increased nutritional demand and larger volume of vegetation ingested.”

May combine two claims.

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 Indian River Lagoon, but also elsewhere.

“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.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Manatees are increasingly at risk from compounding environmental stressors that together pose uncertainties for the long-term stability of this threatened species.

“Manatees are increasingly at risk from compounding environmental stressors that together pose uncertainties for the long-term stability of this threatened species.”