Epidemiological aspects and diagnosis of botulism in waterfowlABSTRACT
Clostridium botulinum is a strictly anaerobic spore-forming bacterium found in soil and sediments, producing botulinum neurotoxins that cause botulism. Type C botulism spores are widely present in wetland sediments and can even be found in tissues of the inhabitants of these environments. The spores germinate in conditions of a lack of oxygen, high temperatures, and the presence of a decomposing organic source, in addition to the presence of a bacteriophage that carries the gene for toxin production. Botulism in waterfowl is mainly caused by C. botulinum type C, and the standard laboratory diagnosis for toxin detection is serum neutralization in mice. However, we tested the application of polymerase chain reaction for the detection and typing of C. botulinum type C with success in three samples from birds.
Keywords
Clostridium botulinum
; polymerase chain reaction; bioassay
The present study not only aimed to present the application of laboratory diagnosis in an outbreak of botulism in aquatic birds but also underscores the crucial role of preventive measures in controlling and preventing avian botulism. By understanding the epidemiological aspects of this disease, we can better implement these measures and protect our avian populations.
Botulism is a neuroparalytic disease resulting from the action of a potent toxin produced by Clostridium botulinum. It is characterized by descending flaccid paralysis, a consequence of the toxin binding to the cholinergic nerve endings, inhibiting acetylcholine release at the neuromuscular junction (Hill et al., 2010).
Clostridium botulinum is an oxygen-intolerant anaerobic bacterium that persists in the environment as dormant spores when environmental conditions are adverse, and these spores are resistant to heat and drying, and remain viable in the environment for years. Type C botulinum spores are widely present in wetland sediments. They can even be found in the tissues of wetland inhabitants, including aquatic insects, mollusks, crustaceans, and many vertebrates, including healthy birds (Biological Resources Division, 1999).
The necessary conditions for spore germination and toxin production include: lack of oxygen, high temperatures, and an organic source of nutrients, usually in the form of dead vertebrates or invertebrates or decaying vegetation, in addition to the presence of a bacteriophage that carries the gene for toxin production (Biological Resources Division, 1999; Grenda et al., 2014).
In humans, botulism is generally associated with toxins of types A, B, E, and F, and four distinct forms of botulism can occur, depending on the route of acquisition of the toxin: foodborne botulism, resulting from the ingestion of the toxin preformed in food; wound botulism, caused by the multiplication of the organism and production of the toxin in a contaminated wound; infant botulism, due to the endogenous production of the toxin by germinated spores of C. botulinum in the immature intestine; and botulism by intestinal colonization in children over 1 year old, or adults, in which no food vehicle was identified, with no evidence of a contaminated wound, but in which there was the possibility of intestinal colonization (CDC, 1998).
Animal botulism is mainly associated with types C and D, although cases of botulism by types A, B, and E have been reported. Mammals, such as cattle, horses, small ruminants, and domestic and aquatic birds, are the most involved in botulism outbreaks (Anniballi et al., 2013).
In aquatic birds, botulism causes mortality in several areas of the world, and its etiology is well known. Botulism in these species is caused by the potent neurotoxin produced by the bacterium C. botulinum type C, and they are unable to sustain flight at the onset of intoxication. However, this sign does not distinguish it from other conditions (Wobeser, 1997; Grenda; Kwiatek, 2009). Ducks with botulism cannot fly, and their legs are paralyzed, so they usually propel themselves across water and mud flats with their wings, while animals with lead poisoning retain the ability to walk and run, although flying becomes more difficult. Later, there are two most easily recognized symptoms: paralysis of the inner eyelid and paralysis of neck muscles, resulting in the inability to hold the head upright, which can lead to drowning (Biological Resources Division, 1999).
Animals in a muddy environment frequently ingest spores. When an animal dies for any other reason, during putrefaction, the tissues are invaded by C. botulinum from the gastrointestinal tract, and toxin production occurs. Larvae of flies and other invertebrates feeding on vertebrate carcasses may contain large amounts of toxins and C. botulinum bacterial cells and spores (Wobeser, 1997). Reed and Rocke (1992) demonstrated the presence of toxic fly larvae both in the carcasses of euthanized wild ducks (control group) and in the carcasses of ducks that had died from botulism, supporting the hypothesis that the formation of toxin postmortem promotes the carcass-larva botulism cycle.
Hubálek and Halouzka (1991) analyzed several samples from a bird mortality site caused by C. botulinum type C in Europe, and the toxin was detected in high concentrations in swan carcasses and necrophagous blowfly larvae and pupae. The toxin was also detected in other invertebrates associated with these carcasses in lower concentrations and occasionally in water samples close to the carcasses. The toxin was not detected in water, larvae, or invertebrate samples collected at a distance greater than 5 meters from the carcasses. Furthermore, the authors kept the larvae with high toxin concentrations in a marshy environment for 131 days. Although there was a decrease in toxin activity (25 to 40 times), it remained very high. Animals containing C. botulinum type C spores in their tissues continually die from many causes in humid areas, and toxin production will occur in some of these carcasses. Each carcass may contain thousands of fly larvae, and a single larva may be enough to poison a small bird (Wobeser, 1997).
The diagnosis of avian botulism is generally based on clinical signs, which are indicative but not specific. Therefore, laboratory investigation must confirm the clinical suspicion and establish a definitive diagnosis. Laboratory tests generally seek to demonstrate the presence of toxins or toxin-producing clostridia in samples collected from symptomatic animals, food, or the environment of the sick animal (Le Maréchal et al., 2017).
The mouse bioassay is still the gold standard test for confirming botulism by detecting the toxin. However, alternative tests to the biological test that are fast, specific, and reliable are increasingly needed, so that there is no longer a need to use experimental animals for diagnosis. The bioassay is based on the intraperitoneal injection of mice with the supernatant obtained after centrifugation of the sample extract and observation of symptoms for 96 hours (CDC, 1998; Grenda et al., 2014). Confirmation of botulinum toxin requires that the laboratory have access to specific antitoxins, which, in addition to the biological risk due to the extreme potency of botulinum neurotoxin, demand strict biosafety measures in the laboratory.
Another methodology that allows the direct detection of C. botulinum is based on molecular biology, since isolating clostridia is difficult (Grenda; Kwiatek, 2009). Polymerase chain reaction (PCR) and Southern blot hybridization are the most commonly described methods, and both techniques have high sensitivity and specificity. C. botulinum DNA can be detected directly in the sample (without pre-enrichment) or at different stages of culture (Grenda et al., 2014).
Following this line, Le Maréchal et al. (2016) tested pooled samples of intestinal contents, liver, spleen, and cloacal swabs in 63 suspected avian botulism outbreaks using real-time PCR after enrichment to elucidate the diagnosis. In 41% (26) of the cases, the diagnosis of botulism was excluded, and in three of them, ionophore poisoning (used to prevent and treat coccidiosis) was diagnosed, whose symptoms are very similar to those of botulism, such as absence of lesions at necropsy, weakness in the legs, paralysis, and high mortality. The liver sample was the most relevant matrix in the laboratory confirmation of avian botulism using PCR, highlighting the need to analyze samples from several animals for a more reliable and robust diagnosis. There was a failure to confirm botulism in intestinal contents or cloacal swabs due to the intestinal microbiota’s inhibition of C. botulinum growth during the enrichment step. Furthermore, the intestinal microbiota can also degrade botulinum toxin, interfering with the success of the biological test.
Given the above, the present study reports the confirmation of botulism in aquatic birds from a park in São Paulo (SP), Brazil.
We conducted a laboratory diagnosis of a botulism outbreak in aquatic birds at a park in São Paulo. In addition to the standard biological test for detecting botulinum toxin (CDC, 1998), we utilized a molecular PCR assay with degenerate primers to detect C. botulinum DNA. This assay specifically targeted the non-toxin-non-hemagglutinin gene, which is highly conserved across all types of C. botulinum, following the methodology described by Hill et al. (2010). Both tests were applied to pooled samples (per bird) of gastric contents and liver from three birds suspected of having botulism. DNA extraction was performed directly from the suspension of samples in phosphate-buffered gelatin used for the biological test, employing the ReliaPrep commercial kit (Promega).
After performing the screening PCR reaction to detect C. botulinum, we differentiated the group III toxinotypes (types C and D) using the methodology described by Takeshi et al. (1996). The primer set targeted the gene region responsible for producing the botulinum toxin light chain in this approach.
The Ethics Committee on the Use of Animals of the Instituto Biológico evaluated and approved the study, registered under protocol CETEA-IB 188/24.
The laboratory diagnosis of botulism in domestic or aquatic birds is based on the identification of the toxin in the blood serum of sick animals and in samples of gastric contents and liver of dead animals.
The biological test detected botulinum toxin type C in the pool samples (gastric contents and liver) of the three birds tested. Likewise, the results of the PCR showed the presence of C. botulinum type C DNA in the same samples. The application of the molecular tool has been increasingly used in the diagnosis of botulism due to the great difficulty in successfully isolating the microorganism, as it is a highly sensitive, specific, and quick-to-perform technique (Grenda; Kwiatek, 2009; Grenda et al., 2014; Chellapandi; Prisilla, 2018; Masters; Palmer, 2021). Our report showed high concordance between the bioassay and PCR techniques. Still, it is necessary to evaluate a larger number of samples to validate the molecular tests and, thus, assess their robustness. The molecular proposal described is fast and straightforward, not requiring a high investment in equipment and reagents.
Because C. botulinum spores and phages that carry the toxin gene are so prevalent in humid environments, they should not be considered a limiting factor in the occurrence of outbreaks in aquatic birds. Other factors may be more critical, such as optimal environmental conditions for spore germination and bacterial growth (high temperatures 30–40°C), available organic material or substrate (since the bacteria require a high-protein substrate for their multiplication) and a means for the toxin to be transferred to the birds, such as invertebrates that feed on organic matter and end up concentrating the toxin (Biological Resources Division, 1999).
For avian botulism control, it is essential that every carcass, regardless of the death cause, be considered as a potential source of botulinum toxin (Reed; Rocke, 1992). One measure to control avian botulism is removing oxygen-deficient environments like those in floating algae clusters. Failure to remove and dispose of carcasses properly also contributes to the presence of organic matter and highly toxic larvae (Wobeser, 1997; Chellapandi; Prisilla, 2018), which are necessary for the spread of poisoning.
In summary, here are some actions that can mitigate the conditions that favor botulism outbreaks in waterfowl:
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Reduction of organic inputs (e.g., sewage, pollutants) in humid environments, particularly in hot weather. The inputs will introduce large amounts of decomposing matter, which can cause the death of aquatic life (which forms a nutrient source for the bacteria);
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Oxygenate the water, either by improving water flow or by pumping;
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Rapid and careful collection of carcasses, especially during outbreak-prone seasons (to remove sources of bacteria);
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Sending biological samples (contents of the gastrointestinal tract and liver in the case of dead birds or blood serum in live birds) to the laboratory for diagnosis;
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Constant monitoring of dead or sick birds;
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In areas of migratory waterfowl, avoid flooding areas where the land has been dried for an extended period, and also avoid lowering water levels during hot weather (both situations can result in the death of fish and aquatic invertebrates, whose carcasses can become a substrate for bacterial growth).
Avian botulism is a disease with characteristic epidemiological aspects that can be used to implement preventive measures in disease outbreaks. The bioassay for the detection of botulinum toxin involves bioethical elements and requires the use of specific antitoxins for its identification; the successful isolation and identification of C. botulinum is tedious, limited, and time-consuming, requiring many days for its completion. The study presented an option for a simple molecular tool (PCR) to directly detect C. botulinum, which can assist in the definitive and rapid determination of the diagnosis.
ACKNOWLEDGEMENTS
Not applicable.
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Peer Review History: Double-blind Peer Review.
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FUNDING
Not applicable.
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ETHICAL APPROVAL
Comissão de Ética no Uso de Animais of Instituto Biológico, under protocol number 189/24.
AVAILABILITY OF DATA AND MATERIAL
All data generated or analyzed during this study are included in this published article.
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Edited by
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Associate Editor:
Silvia Galleti https://orcid.org/0000-0002-0745-5716
