U.S. Geological Survey Techniques and MethodsTechniques and MethodsTMU.S. Department of the InteriorU.S. Geological Survey2328-70472328-705519-E1
10.3133/tm19E1
Avian Botulism Case Definition for WildlifeAvian botulism case definition for wildlifeAvian Botulism Case Definition for WildlifePrepared in cooperation with the Canadian Wildlife Health CooperativeByJulia S.Lankton1 and BrianStevens2
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Acknowledgments
The U.S. Geological Survey National Wildlife Health Center and Canadian Wildlife Health Cooperative Case Definition Joint Working Group would like to thank the staff and pathologists in both organizations, as well as our students and colleagues, for the generous contribution of their collective knowledge, expertise, and time creating this case definition.
Conversion Factors
Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: °F = (1.8 × °C) + 32.
AbbreviationsPCR
polymerase chain reaction
Introduction
Diagnostic laboratories receive carcasses and samples for diagnostic evaluation and pathogen/toxin detection. The intent of a case definition is to provide scientifically based criteria for determining (1) if an individual carcass has a specific disease and the confidence of that diagnosis; and (2) if a pathogen or toxin is evident in a carcass or sample (for example, swab, tissue sample, skin scraping, blood/serum sample, environmental sample, or other). Using these criteria, cases diagnosed with a specific disease (diagnosing disease) will be classified as “confirmed,” “presumptive,” or “suspected;” and evidence of a pathogen or toxin (detecting pathogen/toxin) will be classified as “exposed” or “present/detected.” Classification is based on a combination of factors: individual, place, time, history, clinical signs, diagnostic observations, and (or) diagnostic test results. Case definitions can bring clarity and consistency to the evaluation process. Their use within and between organizations allows more uniform reporting of diseases and etiologic agents.
Case definitions are proposed for use in wildlife diagnostic laboratories and are not intended to replace regulatory standards provided by Government reporting agencies. Ideally, case definitions would be updated periodically as new information becomes available and new test methods are developed. Refer to the glossary for terminology definitions.
Disease/condition.—Avian botulism
Pathogen/toxin etiologic agent(s).—Clostridium botulinum (van Ermengem, 1896; Bergey and others, 1923) toxin types C or E
Scope of the Case Definition
This case definition applies to all avian species.
Case Definition Criteria
The case definition criteria are a concise summary of the current science regarding the clinical signs, history, gross and microscopic observations, and laboratory test results associated with a specific disease or pathogen. Various combinations of the criteria result in different case classifications representing the degree of certainty of the diagnosis.
Individual, Place, and Time Criteria for Diagnosis and Testing
Individual.—Likely all avian species, with the exception of vultures, are susceptible to intoxication; notably, increased index of suspicion exists in waterfowl, shorebirds, and gulls for type C, and fish-eating birds such as loons and gulls for type E botulism (Rocke and Bollinger, 2007).
Place.—No restrictions but increased index of suspicion exists in water with eutrophication, decaying vegetation, or decomposing carcasses (Rocke and Friend, 1999; Rocke and Bollinger, 2007).
Time.—No restrictions, but increased index of suspicion exists during summer and early fall. C. botulinum grows best under anaerobic conditions at 9 degrees Celsius (type E) and 40 degrees Celsius (type C) (Rocke and Friend, 1999; Rocke and Bollinger, 2007).
Field Criteria for Diagnosis
History and clinical signs.—Ingestion of C. botulinum toxin results in an intoxication of variable severity. Diagnostically compatible illness may present with neurological impairments such as the inability to fly, prolapsed third eyelid, and symmetric paralysis in legs and neck that can rapidly progress to death in severe cases. Healthy, sick, and dead birds are commonly observed in the same area. The presence of carcasses can promote the likelihood of botulism spreading through the carcass-maggot cycle for type C (Rocke and Friend, 1999; Rocke and Bollinger, 2007).
Other.—Not applicable.
Laboratory Criteria for Diagnosis
Gross examination.—Diagnostically compatible findings may include good body condition and lack of specific lesions, although body condition can be variable. Signs of drowning or aspiration pneumonia may be evident. Maggots may be found in the gastrointestinal tract for type C (Rocke and Friend, 1999; Rocke and Bollinger, 2007).
Histopathology.—No required lesions.
Diagnostic test(s).—Mouse neutralization test (Lindström and Korkeala, 2006), or polymerase chain reaction (PCR) (Le Maréchal and others, 2017).
Laboratory Criteria Categorization
Laboratory confirmed.—A diagnostically compatible case with a positive mouse neutralization test result of C. botulinum toxin in serum from heart blood collected at necropsy in birds that are freshly dead or were moribund at the time of collection (Lindström and Korkeala, 2006).
Laboratory supportive.—A diagnostically compatible case without a positive mouse neutralization test result; or a diagnostically compatible case with a positive PCR result for C. botulinum toxin genes (Rocke and Friend, 1999; Rocke and Bollinger, 2007).
Exposed.—Not applicable.
Present/detected.—Not applicable.
Supplemental Diagnostic Information
Additional diagnostic comments.—Toxin type should be recorded in etiology. Birds that were euthanized early in the disease process are less desirable for testing and could lead to equivocal results. Birds that were euthanized with barbiturates are not desirable for testing (traces of the barbiturate remaining in the serum may affect the results of the mouse neutralization test by killing the mice). A positive botulism test from a decomposed carcass (because botulism toxin can be produced after death) or instances in which the protected and unprotected mice both become sick or die are inconclusive findings that do not meet the case classification thresholds for this disease/pathogen (Rocke and Bollinger, 2007).
Notifiable/reportable disease.—Not applicable.
Epidemiologic Linkage Criteria for Diagnosis
An epidemiologic linkage can be established by close geographic and temporal proximity (in other words, part of the same mortality event) as one or more confirmed cases of avian botulism or at a site with a recent history of confirmed avian botulism with similar presentation as described in the “Case Definition Criteria” sections.
Case Classification
The sum of the criteria listed in the “Case Definition Criteria” sections (individual, place, time, field, laboratory, and epidemiologic linkage criteria) associated with a particular disease or pathogen/toxin in an individual animal or specimen add up to a case classification (fig. 1; table 1).
Case definition criteria add up to the case classifications. From Miller and others (2024).
Figure 1. Diagram showing case definition criteria add up to the case classifications.
Individual, place, and time, field, laboratory, and epidemiological linkage add up to a classification.
Depending on the confidence in the results, cases of a specific disease will be classified as “confirmed,” “presumptive,” or “suspected;” and evidence of a pathogen or toxin will be classified as “exposed” or “present/detected” (table 1; refer to glossary for definitions). A specific case classification may have more than one pathway to it. Not all classifications may be used for every disease. Although an epidemiological linkage may be present in all classifications, the “suspected” case classification requires compatible epidemiological linkage criteria are met. Individual disease and pathogen specific details are presented in table 1.
Note.—The field and laboratory criteria in table 1 reflect the typical presentation of avian botulism. The exact presentation in an individual animal or specimen may vary from what is presented in table 1 but would still conform with the information presented in the “Field Criteria for Diagnosis” and “Laboratory Criteria for Diagnosis” sections.
Case classification chart for avian botulism and <italic>Clostridium botulinum</italic> toxin types C and E.
Table 1. Case classification chart for avian botulism and Clostridium botulinum toxin types C and E.
[The detecting toxin/pathogen case classifications (exposed and present/detected) are not applicable to this case definition]
Individual, place, and time criteria
Field criteria (if available)
Laboratory criteria
Epidemiological linkage criteria
Classification
Diagnosing Avian botulism
Avian, any place, anytime
Paralysis and good body condition (If bird is found dead then may not have any field criteria information.)
Meets laboratory confirmed criteria:Gross examination.—No lesionsandHistopathology.—NoneandDiagnostic test(s).—Positive mouse neutralization from moribund or freshly dead bird
Optional: Close geographic or temporal proximity to a confirmed case, or at a site with a recent history of confirmed avian botulism
Confirmed
Avian, any place, anytime
Paralysis and good body condition (If bird is found dead then may not have any field criteria information.)
Meets laboratory supportive criteria:Gross examination.—No lesionsandHistopathology.—NoneandDiagnostic test(s).— Equivocal mouse neutralization test result and (or) positive PCR results for C. botulinum toxin genes
Optional: Close geographic or temporal proximity to a confirmed case, or at a site with a recent history of confirmed avian botulism
Presumptive
Avian, any place, anytime
Paralysis and good body condition (If bird is found dead then may not have any field criteria information.)
Some information but not enough to meet laboratory supportive criteria:Gross examination.—No lesionsandHistopathology.—NoneandDiagnostic test(s).— No test results (test not performed) or test is negative
Required: Close geographic or temporal proximity to a confirmed case, or at a site with a recent history of confirmed avian botulism
Suspected
Quality-Assurance Review Schedule
The Canadian Wildlife Health Cooperative and the U.S. Geological Survey National Wildlife Health Center staff plan to review this case definition periodically to incorporate new scientific information and test methods as needed.
Planned date for next review.—June 1, 2025
Schedule.—June 2025 and then every 3–5 years—or sooner if science about avian botulism changes substantially.
Impact
Applying case definitions in diagnostic, surveillance, and research efforts can help standardize data, making it easier to understand and analyze within and between diagnosticians and laboratories. Laboratories are encouraged to store the case classification assigned to each specimen or sample in their data system so that it can be readily and reliably retrievable.
References CitedLe Maréchal, C., Rouxel, S., Ballan, V., Houard, E., Poezevara, T., Bayon-Auboyer, M., Souillard, R., Morvan, H., Baudouard, M., Woudstra, C., Mazuet, C., Le Bouquin, S., Fach, P., Popoff, M., and Chemaly, M., 2017, Development and validation of a new reliable method for the diagnosis of avian botulism: PLoS One, v. 12, no. 1, p. e0169640. [Also available at https://doi.org/10.1371/journal.pone.0169640.]Lindström, M., and Korkeala, H., 2006, Laboratory diagnostics of botulism: Clinical Microbiology Reviews, v. 19, no. 2, p. 298–314. [Also available at https://doi.org/10.1128/CMR.19.2.298-314.2006.]Miller, K.J.G., Parmley, E.J., Ballmann, A., Buckner, J., Jones, M., Lankton, J.S., Zimmer, M., and Lankau, E., 2024, [Disease/condition] case definition [template] for wildlife: U.S. Geological Survey Techniques and Methods, book 19, chap. A1, 8 p., https://doi.org/10.3133/tm19A1.Rocke, T.E., and Bollinger, T.K., 2007, Avian botulism, in Thomas, N.J., Hunter, D.B., and Atkinson, C.T., eds., Infectious diseases of wild birds: Oxford, Blackwell Publishing, p. 377–416. [Also available at https://doi.org/10.1002/9780470344668.ch21.]Rocke, T., and Friend, M., 1999, Avian botulism in Field manual of wildlife diseases—General field procedures and diseases of birds: U.S. Geological Survey Information and Technology Report 1999–0001, p. 271–282. [Also available at https://pubs.usgs.gov/publication/2001150.]Glossaryadditional diagnostic comments
Any additional diagnostic notes pertinent to recording/reporting (for example, requests for strain/serovar/variant reporting, inconclusive/ambiguous results, or “not applicable”).
case classification
The sum of the factors in the “Case Definition Criteria” sections of the case definition including individual (for example, species, age group), place, time, history, clinical signs, diagnostic observations, and (or) diagnostic test results, associated with a particular disease or pathogen/toxin in an individual animal or specimen. Depending on the confidence in the results, cases of a specific disease will be classified as “confirmed,” “presumptive,” or “suspected;” and a pathogen or toxin will be classified as “exposed” or “present/detected.”
case definition
A consistently applied, scientifically based and clearly defined set of field, gross, histopathology, laboratory, or epidemiologic criteria used to classify an individual animal or sample to a specific disease or pathogen/toxin for surveillance or outbreak reporting purposes (based on the combination of the criteria and confidence in the results).
confirmed case
The combination of individual (for example, species, age group), place, time, history, clinical signs, and laboratory criteria for diagnosis with the highest level of certainty for accepted diagnostic testing as stated in the case definition. Example: Cardinal with clinical signs, gross and microscopic lesions compatible with salmonellosis, and positive bacterial culture for Salmonella enterica enterica in the liver.
diagnostic test(s)
Laboratory tests typically used to determine this diagnosis or detect the pathogen/toxin; for example, bacterial culture.
diagnostically compatible
An animal that meets the individual (for example, species, age group), place, time, field, and laboratory criteria for a particular disease as stated in the case definition.
disease
Any disorder of structure or function that may produce specific clinical signs; disease can be infectious or noninfectious.
disease agent
Any pathogen, toxin, or other known cause of disease.
epidemiologically linked
A case that has temporal, geographic, or other relevant linkages to one or more confirmed cases as described under “Epidemiologic Linkage Criteria for Diagnosis” in the case definition.
exposed
Detection of a toxin in tissues or body fluids at a concentration above acceptable background levels but below the documented lethal threshold level for the species. This may apply to a toxin detected in the absence of documented lethal threshold levels. This category can also include serological evidence of infection in the absence of other information such as organism detection or disease diagnosis.
gross examination
Gross necropsy observations in a carcass or sample that are diagnostically compatible with disease.
histopathology
General histopathology observations in a carcass or sample that are diagnostically compatible with disease.
history and clinical signs
Field observations and changes to behavior, appearance, or abilities in live animals/populations that are diagnostically compatible with disease. Photograph or video evidence may be used when appropriate.
individual
The common age groups, species, or other characteristics that increase disease or pathogen/toxin suspicion.
laboratory confirmed
The strongest degree of assurance in identification of a disease agent of interest and evidence of the associated disease based on one or more accepted laboratory methods. A test or combination of methods that has been scientifically accepted as definitive for a particular disease agent and the associated disease. Example: Positive bacterial isolation for salmonella plus compatible gross and histologic lesions for salmonellosis.
laboratory criteria for diagnosis
The gross, microscopic, molecular, culture, analytical or other laboratory test criteria used to determine the presence of a specific disease agent and evidence of the disease itself. These are categorized based on the validity and performance of the test(s). Categories are “laboratory confirmed,” “laboratory supportive,” “exposed,” and “present/detected.” Where possible, references for the current accepted science for a given disease and pathogen are provided in the case definition. For some select new or emerging diseases the laboratory criteria may be based on the collective expertise of pathologists at the U.S. Geological Survey National Wildlife Health Center and the Canadian Wildlife Disease Cooperative or other institutions.
laboratory supportive
Laboratory results that are less than definitive for a specific disease agent and the associated disease. A test or combination or methods whose results support the diagnosis or a particular disease but are not considered definitive; for example, a screening test. Test result interpretation may be based on the tissue tested (for example, culture of amphibian skin surface versus internal tissue) or postmortem condition of the sample. Example: Gross and histologic lesions compatible with salmonellosis (without laboratory testing).
notifiable/reportable disease
A disease or pathogen that by law must be disclosed to State, Provincial, and (or) Federal agricultural or public health authorities.
other (field criteria)
Additional pertinent comments about presentation (for example, potential for carrier status).
place
Locations and other geographic features that increase disease or pathogen/toxin suspicion.
present/detected
Laboratory detection of a potentially pathogenic agent in the absence of findings diagnostically compatible with the associated disease. Often used when tracking a known or suspected asymptomatic carrier state (for example, Salmonella or duck virus enteritis) or when documenting detection of an agent that is of increased diagnostic or epidemiologic interest, even in the absence of evidence of illness (for example, new or emerging disease or syndrome).
presumptive case
The combination of individual (for example, species, age group), place, time, history, clinical signs and laboratory criteria for diagnosis that has a moderate degree of certainty as stated in the case definition. This uncertainty may be due to the test performed, postmortem decomposition of the carcass affecting observation or interpretation of gross and or histopathologic lesions, inadequate sample for testing due to scavenging or carcass size, inconclusive test results, or lack of a definitive diagnostic test. Enough information is available to conclude the disease is most likely present but not enough information available to conclude the disease is definitively present. Example: Raccoon with compatible histologic lesions for parvovirus without additional laboratory test results.
scope
Indicates what species, when and (or) where this protocol applies; for example, specifics regarding the disease agent, animal class, sex, age group, location, season, antemortem or postmortem sample collection, environmental samples, and so on.
suspected case
This is primarily based on a combination of individual, place, time, minimal or nonspecific field and laboratory information and a geographic and temporal (epidemiologic) connection to a confirmed case. Not enough information is available to meet the threshold in the case definition for a confirmed or presumptive case, but the diagnosis can reasonably be inferred by the close association with confirmed cases of a particular disease in other animals collected from the same general location and time. Example: A specimen with a geographic or temporal link to a confirmed case of a disease that is not tested but was examined and may have nonspecific gross or histopathologic findings that are compatible with that disease.
time
The season(s), months, or other temporal factors that increase disease or pathogen/toxin suspicion.
wildlife
Free ranging vertebrate species (mammals, birds, reptiles, amphibians, and fish).
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