Clostridium botulinum

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Clostridium botulinum
Clostridium botulinum 01.png
Clostridium botulinum stained with gentian violet.
Scientific classification
Domain: Bacteria
Phylum: Firmicutes
Class: Clostridia
Order: Clostridiales
Family: Clostridiaceae
Genus: Clostridium
Species: C. botulinum
Binomial name
Clostridium botulinum
van Ermengem, 1896

Clostridium botulinum is a Gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium with the ability to produce a neurotoxin known as botulinum. [1][2]

The botulinum toxin can cause a severe flaccid paralytic disease in humans and other animals[2] and is the most potent toxin known to humankind, natural or synthetic, with a lethal dose of 1.3–2.1 ng/kg in humans.[3]

C. botulinum is a diverse group of pathogenic bacteria initially grouped together by their ability to produce botulinum toxin and now known as four distinct groups, C. botulinum groups I-IV. C. botulinum groups I-IV, as well as some strains of Clostridium butyricum and Clostridium baratii, are the bacteria responsible for producing botulinum toxin.[1]

C. botulinum is responsible for foodborne botulism (ingestion of preformed toxin), infant botulism (intestinal infection with toxin-forming C. botulinum), and wound botulism (infection of a wound with C. botulinum). C. botulinum produces heat-resistant endospores that are commonly found in soil and are able to survive under adverse conditions.[1]

C. botulinum is commonly associated with bulging canned food; bulging, misshapen cans are due to an internal increase in pressure caused by gas produced by the bacteria.[4]

Microbiology[edit]

C. botulinum is a Gram-positive, rod-shaped, spore-forming bacterium. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme superoxide dismutase, which is an important antioxidant defense in nearly all cells exposed to oxygen.[5] C. botulinum is only able to produce the neurotoxin during sporulation, which can only happen in an anaerobic environment. Other bacterial species produce spores in an unfavorable growth environment to preserve the organism's viability and permit survival in a dormant state until the spores are exposed to favorable conditions.

C. botulinum is divided into four distinct phenotypic groups (I-IV) and is also classified into seven serotypes (A-G) based on the antigenicity of the botulinum toxin produced.[6][7]

Groups[edit]

The classification into groups is based on the ability of the organism to digest complex proteins.[8][9] Studies at the DNA and rRNA level support the subdivision of the species into groups I-IV. Most outbreaks of human botulism are caused by group I (proteolytic) or II (non-proteolytic) C. botulinum. Group III organisms mainly cause diseases in animals. Group IV C. botulinum has not been shown to cause human or animal disease.

Botulinum toxin[edit]

Neurotoxin production is the unifying feature of the species. Seven types of toxins have been identified that are allocated a letter (A-G). All toxins are rapidly destroyed at 100 °C, but they are resistant to degradation by enzymes found in the gastrointestinal tract. This allows for ingested toxin to be absorbed from the intestines into the bloodstream.[3]

Most strains produce one type of neurotoxin, but strains producing multiple toxins have been described. C. botulinum producing B and F toxin types have been isolated from human botulism cases in New Mexico and California.[10] The toxin type has been designated Bf as the type B toxin was found in excess to the type F. Similarly, strains producing Ab and Af toxins have been reported. Evidence indicates the neurotoxin genes have been the subject of horizontal gene transfer, possibly from a viral source. This theory is supported by the presence of integration sites flanking the toxin in some strains of C. botulinum. However, these integrations sites are degraded, indicating that the C. botulinum acquired the toxin genes quite far in the evolutionary past.

Botulinum toxin types[edit]

Only botulinum toxin types A, B, E, and F cause disease in humans. Types A, B, and E are associated with foodborne illness, with type E specifically associated with fish products. Type C produces limberneck in birds and type D causes botulism in other mammals. No disease is associated with type G.[11] The "gold standard" for determining toxin type is a mouse bioassay, but the genes for types A, B, E, and F can now be readily differentiated using quantitative PCR.[12]

A few strains from organisms genetically identified as other Clostridium species have caused human botulism: C. butyricum has produced type E toxin[13] and C. baratii had produced type F toxin.[14][15] The ability of C. botulinum to naturally transfer neurotoxin genes to other clostridia is concerning, especially in the food industry, where preservation systems are designed to destroy or inhibit only C. botulinum but not other Clostridium species.

Phenotypic groups of Clostridium botulinum

Properties

Group I

Group II

Group III

Group IV

Toxin Types

A, B, F B, E, F C, D G

Proteolysis

+ weak

Saccharolysis

+

Disease host

human human animal

Toxin gene

chromosome/plasmid chromosome/plasmid bacteriophage plasmid

Close relatives

C. sporogenes, C. putrificum C. butyricum, C. beijerinickii C. haemolyticum, C. novyi type A C. subterminale, C. haemolyticum

Laboratory isolation[edit]

In the laboratory, C. botulinum is usually isolated in tryptose sulfite cycloserine (TSC) growth medium in an anaerobic environment with less than 2% oxygen. This can be achieved by several commercial kits that use a chemical reaction to replace O2 with CO2. C. botulinum is a lipase-positive microorganism that grows between pH of 4.8 and 7.0 and cannot use lactose as a primary carbon source, characteristics important for biochemical identification.[16]

Taxonomy history[edit]

C. botulinum was first recognized and isolated in 1895 by Emile van Ermengem from home-cured ham implicated in a botulism outbreak.[17] The isolate was originally named Bacillus botulinus, after the Latin word for sausage, botulus. ("Sausage poisoning" was a common problem in 18th- and 19th-century Germany, and was most likely caused by botulism)[18] However, isolates from subsequent outbreaks were always found to be anaerobic spore formers, so Ida A. Bengtson proposed that the organism be placed into the genus Clostridium, as the Bacillus genus was restricted to aerobic spore-forming rods.[19]

Since 1959, all species producing the botulinum neurotoxins (types A-G) have been designated C. botulinum. Substantial phenotypic and genotypic evidence exists to demonstrate heterogeneity within the species. This has led to the reclassification of C. botulinum type G strains as a new species, C. argentinense.[20]

Group I C. botulinum strains that do not produce a botulin toxin are referred to as C. sporogenes.[21]

The complete genome of C. botulinum has been sequenced at Wellcome Trust Sanger Institute in 2007.[22]

Pathology[edit]

Botulism poisoning can occur due to preserved or home-canned, low-acid food that was not processed using correct preservation times and/or pressure.

Foodborne botulism "Signs and symptoms of foodborne botulism typically begin between 18 and 36 hours after the toxin gets into your body, but can range from a few hours to several days, depending on the amount of toxin ingested."[23]

  • Double vision
  • Blurred vision
  • Drooping eyelid
  • Nausea, vomiting, and abdominal cramps
  • Slurred speech
  • Trouble breathing
  • Difficulty in swallowing
  • Dry mouth
  • Muscle weakness
  • Constipation
  • Reduced or absent deep tendon reactions, such as in the knee.

Wound botulism Most people who develop wound botulism inject drugs several times a day, so it's difficult to determine how long it takes for signs and symptoms to develop after the toxin enters the body. Most common in people who inject black tar heroin, wound botulism signs and symptoms include:[23]

  • Difficulty swallowing or speaking
  • Facial weakness on both sides of the face
  • Blurred or double vision
  • Drooping eyelids
  • Trouble breathing
  • Paralysis

Infant botulism If infant botulism is related to food, such as honey, problems generally begin within 18 to 36 hours after the toxin enters the baby's body. Signs and symptoms include:

  • Constipation (often the first sign)
  • Floppy movements due to muscle weakness and trouble controlling the head
  • Weak cry
  • Irritability
  • Drooling
  • Drooping eyelids
  • Tiredness
  • Difficulty sucking or feeding
  • Paralysis[23]

Beneficial effects of botulinum toxin: Purified botulinum toxin is diluted by a physician for treatment:

  • Congenital pelvic tilt
  • Spasmodic dysphasia (the inability of the muscles of the larynx)
  • Achalasia (esophageal stricture)
  • Strabismus (crossed eyes)
  • Paralysis of the facial muscles
  • Failure of the cervix
  • Blinking frequently
  • Anti-cancer drug delivery[24]

Adult intestinal toxemia: A very rare form of botulism that occurs by the same route as infant botulism but is among adults. Occurs rarely and sporadically. Signs and symptoms include:

  • Abdominal pain
  • Blurred vision
  • Diarrhea
  • Dysarthria
  • Imbalance
  • Weakness in arms and hand area

[25]

C. botulinum in different geographical locations[edit]

A number of quantitative surveys for C. botulinum spores in the environment have suggested a prevalence of specific toxin types in given geographic areas, which remain unexplained.

North America[edit]

Type A C. botulinum predominates the soil samples from the western regions, while type B is the major type found in eastern areas.[26] The type-B organisms were of the proteolytic type I. Sediments from the Great Lakes region were surveyed after outbreaks of botulism among commercially reared fish, and only type E spores were detected.[27][28][29] In a survey, type-A strains were isolated from soils that were neutral to alkaline (average pH 7.5), while type-B strains were isolated from slightly acidic soils (average pH 6.25).

Europe[edit]

C. botulinum type E is prevalent in aquatic sediments in Norway and Sweden,[30] Denmark,[31] the Netherlands, the Baltic coast of Poland, and Russia.[26] The type-E C. botulinum was suggested to be a true Aquatic ecosystem|aquatic organism, which was indicated by the correlation between the level of type-E contamination and flooding of the land with seawater. As the land dried, the level of type E decreased and type B became dominant.

In soil and sediment from the United Kingdom, C. botulinum type B predominates. In general, the incidence is usually lower in soil than in sediment. In Italy, a survey conducted in the vicinity of Rome found a low level of contamination; all strains were proteolytic C. botulinum types A or B.[32]

Australia[edit]

C. botulinum type A was found to be present in soil samples from mountain areas of Victoria.[33] Type-B organisms were detected in marine mud from Tasmania.[34][verification needed] Type-A C. botulinum has been found in Sydney suburbs and types A and B were isolated from urban areas. In a well-defined area of the Darling-Downs region of Queensland, a study showed the prevalence and persistence of C. botulinum type B after many cases of botulism in horses.

Other[edit]

A "mouse protection" or "mouse bioassay" test determines the type of C. botulinum toxin present using monoclonal antibodies. An enzyme-linked immunosorbent assay (ELISA) with digoxigenin-labeled antibodies can also be used to detect the toxin,[35] and quantitative PCR can detect the toxin genes in the organism.[12]

C. botulinum is also used to prepare the medicaments Botox, Dysport, Xeomin, and Neurobloc used to selectively paralyze muscles to temporarily relieve muscle function. It has other "off-label" medical purposes, such as treating severe facial pain, such as that caused by trigeminal neuralgia.

Botulinum toxin produced by C. botulinum is often believed to be a potential bioweapon as it is so potent that it takes about 75 nanograms to kill a person (LD50 of 1 ng/kg,[36] assuming an average person weighs ~75 kg); 1 kilogram of it would be enough to kill the entire human population. For comparative purposes, a quarter of a typical grain of sand's weight (350 ng) of botulinum toxin would constitute a lethal dose for humans.

C. botulinum is a soil bacterium. The spores can survive in most environments and are very hard to kill. They can survive the temperature of boiling water at sea level, thus many foods are canned with a pressurized boil that achieves even higher temperatures, sufficient to kill the spores.

Growth of the bacterium can be prevented by high acidity, high ratio of dissolved sugar, high levels of oxygen, very low levels of moisture, or storage at temperatures below 3 °C (38 °F) for type A. For example, in a low-acid, canned vegetable such as green beans that are not heated enough to kill the spores (i.e., a pressurized environment) may provide an oxygen-free medium for the spores to grow and produce the toxin. However, pickles are sufficiently acidic to prevent growth; even if the spores are present, they pose no danger to the consumer. Honey, corn syrup, and other sweeteners may contain spores, but the spores cannot grow in a highly concentrated sugar solution; however, when a sweetener is diluted in the low-oxygen, low-acid digestive system of an infant, the spores can grow and produce toxin. As soon as infants begin eating solid food, the digestive juices become too acidic for the bacterium to grow.

Growth conditions[edit]

The control of food-borne botulism caused by C. botulinum is based almost entirely on thermal destruction (heating) of the spores or inhibiting spore germination into bacteria and allowing cells to grow and produce toxins in foods. Conditions conducive of growth are dependent on various environmental factors. Growth of C. botulinum is a risk in low acid foods as defined by having a pH greater than 4.6[37] although growth is significantly retarded for pH below 4.9. There have been some cases and specific conditions reported to sustain growth with pH below 4.6.[38][39]

C. botulinum is an obligate anaerobe that is widely distributed in nature and is assumed to be present on all food surfaces. Its optimum growth temperature is within the mesophilic range. In spore form, it is the most heat resistant pathogen that can survive in low acid foods and grow to produce toxin. The toxin attacks the nervous system and will kill an adult at a dose of around 75 ng.[36] This toxin is detoxified by holding food at 100 °C for 10 minutes.

Diagnosis[edit]

Physicians may consider the diagnosis of botulism based on a patient’s clinical presentation, which classically includes an acute onset of bilateral cranial neuropathies and symmetric descending weakness.[40][41] Other key features of botulism include an absence of fever, symmetric neurologic deficits, normal or slow heart rate and normal blood pressure, and no sensory deficits except for blurred vision.[42][43] A careful history and physical examination is paramount in order to diagnose the type of botulism, as well as to rule out other conditions with similar findings, such as Guillain-Barre syndrome, stroke, and myasthenia gravis. Depending on the type of botulism considered, different tests for diagnosis may be indicated.

Foodborne Botulism: serum analysis for toxins by bioassay in mice should be done, as the demonstration of the toxins is diagnostic.[44]

Wound Botulism: isolation of C. botulinum from the wound site should be attempted, as growth of the bacteria is diagnostic.[45]

Adult Enteric and Infant Botulism: isolation and growth of C. botulinum from stool samples is diagnostic.[46] Infant botulism is a diagnosis which is often missed in the emergency room.

Other tests that may be helpful in ruling out other conditions are:

Treatments and Vaccines[edit]

In the case of a diagnosis or suspicion of botulism, patients should be hospitalized immediately, even if the diagnosis and/or tests are pending. If botulism is suspected, patients should be treated immediately with antitoxin therapy in order to reduce mortality. Immediate intubation is also highly recommended, as respiratory failure is the primary cause of death from botulism.[50][51][52]

In Canada, there are currently only 3 antitoxin therapies available, which are accessible through Health Canada Special Access Program (SAP).[53] The 3 types of antitoxin therapies are: 1) GlaxoSmithKline trivalent Types ABE, 2) NP-018 (heptavalent) Types A to G, and 3) BabyBIG®, Botulism Immune Globulin Intravenous (Human) (BIG-IV) for pediatric patients under the age of one year.[54]

Outcomes vary between one and three months, but with prompt interventions, mortality from botulism ranges from less than 5 percent to 8 percent.[55]

References[edit]

  1. ^ a b c Peck, MW (2009). "Biologycoat and genomic analysis of Clostridium botulinum". Advances in microbial physiology. Advances in Microbial Physiology. 55: 183–265, 320. doi:10.1016/s0065-2911(09)05503-9. ISBN 978-0-12-374790-7. PMID 19573697. 
  2. ^ a b Lindström, M; Korkeala, H (Apr 2006). "Laboratory diagnostics of botulism". Clinical Microbiology Reviews. 19 (2): 298–314. doi:10.1128/cmr.19.2.298-314.2006. PMC 1471988Freely accessible. PMID 16614251. 
  3. ^ a b (2010). Chapter 29. Clostridium, Peptostreptococcus, Bacteroides, and Other Anaerobes. In Ryan K.J., Ray C (Eds), Sherris Medical Microbiology, 5th ed. ISBN 978-0-07-160402-4
  4. ^ Schneider, Keith R.; Silverberg, Rachael; Chang, Alexandra; Goodrich Schneider, Renée M. (9 January 2015). "Preventing Foodborne Illness: Clostridium botulinum". edis.ifas.ufl.edu. University of Florida IFAS Extension. Retrieved 7 February 2017. 
  5. ^ Doyle, Michael P. (2007). Food Microbiology: Fundamentals and Frontiers. ASM Press. ISBN 1-55581-208-2. 
  6. ^ Peck, MW; Stringer, SC; Carter, AT. (2011). "Clostridium botulinum in the post-genomic era". Food Microbiol. 28 (2): 183–191. doi:10.1016/j.fm.2010.03.005. PMID 21315972. 
  7. ^ Shukla, HD; Sharma, SK. (2005). "Clostridium botulinum: a bug with beauty and weapon". Crit Rev Microbiol. 31 (1): 11–18. doi:10.1080/10408410590912952. PMID 15839401. 
  8. ^ L. V. Holdeman, J. B. Brooks. 1970. Variation among strains of Clostridium botulinum and related clostridia. Protocols of the first U.S-Japan conference on Toxic Microorganisms. pp. 278–286
  9. ^ L. D. S. Smith, G. Hobbs. 1974. Genus III Clostridium Prazmowski 1880, 23. In R. E. Buchanan, N. E. gibbons (eds.), Bergey's Manual of Determinative Bacteriology, 8th ed. William & Wilkins, Baltimore. pp. 551–572. ISBN 978-0-683-00603-2
  10. ^ Hatheway, C. L.; McCroskey, L. M. (1987). "Examination of faeces for diagnosis of infant botulism in 336 patients". J. Clin. Microbiol. 25 (12): 2334–2338. PMC 269483Freely accessible. PMID 3323228. 
  11. ^ (2013). Chapter 11. Spore-Forming Gram-Positive Bacilli: Bacillus and Clostridium Species. In Brooks G.F., Carroll K.C., Butel J.S., Morse S.A., Mietzner T.A. (Eds), Jawetz, Melnick, & Adelberg's Medical Microbiology, 26th ed. ISBN 978-0-07-179031-4
  12. ^ a b Satterfield BA, Stewart AF, Lew CS, Pickett DO, Cohen MN, Moore EA, Luedtke PF, O'Neill KL, Robison RA (2010). "A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F". J. Med. Microbiol. 59 (Pt 1): 55–64. doi:10.1099/jmm.0.012567-0. PMID 19779029. 
  13. ^ Aureli, P.; Fenicia, L.; Pasolini, B.; Gianfrancesche, M.; Mccroskey, J. M.; Hatheway, C. L. (1986). "Two cases of type E infant botulism caused by neurotoxigenic Clostridium botulinum in Italy". J. Infect. Dis. 154 (2): 207–211. doi:10.1093/infdis/154.2.207. PMID 3722863. 
  14. ^ Hall, J. D.; McCroskey, L. M.; Pincomb, B. J.; Hatheway, C. L. (1985). "Isolation of an organism resembling Clostridium baratii which produces a type F botulinal toxin from an infant with botulism". J. Clin. Microbiol. 21 (4): 654–655. PMC 271744Freely accessible. PMID 3988908. 
  15. ^ Notermans, S.; Havellar, A. H. (1980). "Removal and inactivation of botulinum toxin during production of drinking water from surface water". Antonie van Leeuwenhoek. 46 (5): 511–514. doi:10.1007/BF00395840. 
  16. ^ . (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0-13-144329-1. 
  17. ^ E. van Ergmengem. 1897. Über einen neuen anaeroben Bacillus und seine Beziehungen Zum Botulismus. Zentralbl. Hyg. Infektionskr. 26:1–8.
  18. ^ Frank J. Erbguth. Historical notes on botulism, Clostridium botulinum, botulinum toxin, and the idea of the therapeutic use of the toxin. Movement Disorders. Volume 19, Issue S8, pp. S2–S6, March 2004.
  19. ^ I. A. Bengston. 1924. Studies on organisms concerned as causative factors in botulism. Hyg. Lab. Bull. 136:101
  20. ^ J. C. Suen, C. L. Hatheway, A. G. Steigerwalt, D. J. Brenner. 1988, Clostridium argentinense sp.nov.: a genetically homogeneous group composed of all strains of Clostridium botulinum type G and some nontoxigenic strains previously identified as Clostridium subterminale or Clostridium hastiforme. Int. J. Sys. Bacteriol. 38:375–381.
  21. ^ Judicial Commission of the International Committee on Systematic Bacteriology (1999) Rejection of Clostridium putrificum and conservation of Clostridium botulinum and Clostridium sporogenes Opinion 69. International Journal of Systematic Bacteriology. 49:339.
  22. ^ "Clostridium botulinum". Wellcome Trust Sanger Institute. 2007. Retrieved January 25, 2016. 
  23. ^ a b c "Botulism Symptoms". Mayo Clinic. June 13, 2015. Retrieved January 25, 2016. 
  24. ^ Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Hauer J, Layton M, Lillibridge S, Osterholm MT, O'Toole T, Parker G, Perl TM, Russell PK, Swerdlow DL, Tonat K (2001). "Botulinum toxin as a biological weapon: medical and public health management". JAMA. 285 (8): 1059–1070. doi:10.1001/jama.285.8.1059. PMID 11209178. 
  25. ^ "Botulism." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 2016. Web. 23 Oct. 2016.
  26. ^ a b A. H. W. Hauschild. 1989. Clostridium botulinum. In M. P. Doyle (ed.), Food-borne Bacterial Pathogens. Marcel Dekker, New York. pp. 111–189
  27. ^ Bott, T. L.; Johnson, J.; Foster, E. M.; Sugiyama, H. (1968). "Possible origin of the fish incidences of Clostridium botulinum type E in an inland bay (Green Bay of Lake Michigan)". J. Bacteriol. 95: 1542. 
  28. ^ M. W. Eklund, M. E. Peterson, F. T. Poysky, L. W. Peck, J. F. Conrad. 1982. Botulism in juvenile Coho salmon (Onocorhynchus kisutch) in the United States. Aquaculture 27:1–11
  29. ^ M. W. Eklund, F. T. Poysky M. E. Peterson, L. W. Peck, Brunson. 1984. Type E botulism in salmonids and conditions contributing to outbreaks. Aquaculture 41:293–309.
  30. ^ A. Johannsen. 1963. Clostridium botulinum in Sweden and the adjacent waters. J. Appl. Bacteriol. 26:43–47.
  31. ^ Huss, H. H. (1980). "Distribution of Clostridium botulinum". Appl. Environ. Microbiol. 39 (4): 764–769. PMC 291416Freely accessible. PMID 6990867. 
  32. ^ Creti, R.; Fenicia, J.; Aureli, P. (1990). "Occurrence of Clostridium botulinum in the soil of the vicinity of Rome". Curr. Microbiol. 20 (5): 317. doi:10.1007/bf02091912. 
  33. ^ Eales, C. E.; Gillespie, J. M. (1947). "the isolation of Clostridium botulinum type A from Victorian soils. Aust. J". Sci. 10: 20–21. 
  34. ^ Ohye, W. J. Scott (1957). "Studies in the physiology of Clostridium botulinum type E. Aust. L. Biol". Sci. 10: 85–94. 
  35. ^ Shashi K. Sharma, Joseph. L. Ferreira, Brian S. Eblen and Richard C. Whitingand. Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies. Appl. Environ. Microbiol.. February 2006 vol. 72 no. 2 1231–1238. doi:10.1128/AEM.72.2.1231-1238.2006
  36. ^ a b Fleming, Diane O. "Biological Safety: principles and practices". ASM Press. 2000: 267. 
  37. ^ "Guidance for Commercial Processors of Acidified & Low-Acid Canned Foods". U.S. Food and Drug Administration. Retrieved 8 October 2016. 
  38. ^ Lund, Barbara M.; Graham, Ann F.; Franklin, Jeremy G. (June 1987). "The effect of acid pH on the probability of growth of proteolytic strains of Clostridium botulinum". International Journal of Food Microbiology. 4 (3): 215–226. doi:10.1016/0168-1605(87)90039-0. 
  39. ^ Raatjes, G. J. M.; Smelt, J. P. P. M. (4 October 1979). "Clostridium botulinum can grow and form toxin at pH values lower than 4.6". Nature. 281 (5730): 398–399. Bibcode:1979Natur.281..398R. doi:10.1038/281398a0. 
  40. ^ Cherington, M. (June 1998). "Clinical spectrum of botulism". Muscle & Nerve. 21 (6): 701–710. ISSN 0148-639X. PMID 9585323. 
  41. ^ Cai, Shuowei; Singh, Bal Ram; Sharma, Shashi (April 2007). "Botulism diagnostics: from clinical symptoms to in vitro assays". Critical Reviews in Microbiology. 33 (2): 109–125. doi:10.1080/10408410701364562. ISSN 1040-841X. PMID 17558660. 
  42. ^ "Diagnosis and Treatment | Botulism | CDC". www.cdc.gov. Retrieved 2017-10-08. 
  43. ^ "Botulism: Rare but serious food poisoning". Mayo Clinic. Retrieved 2017-11-18. 
  44. ^ Lindström, Miia; Korkeala, Hannu (April 2006). "Laboratory Diagnostics of Botulism". Clinical Microbiology Reviews. 19 (2): 298–314. doi:10.1128/CMR.19.2.298-314.2006. ISSN 0893-8512. PMC 1471988Freely accessible. PMID 16614251. 
  45. ^ Akbulut, D.; Grant, K. A.; McLauchlin, J. (September 2005). "Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments". Journal of Clinical Microbiology. 43 (9): 4342–4348. doi:10.1128/JCM.43.9.4342-4348.2005. ISSN 0095-1137. PMC 1234055Freely accessible. PMID 16145075. 
  46. ^ Dezfulian, M; McCroskey, L M; Hatheway, C L; Dowell, V R (March 1981). "Selective medium for isolation of Clostridium botulinum from human feces". Journal of Clinical Microbiology. 13 (3): 526–531. ISSN 0095-1137. PMC 273826Freely accessible. PMID 7016901. 
  47. ^ O'Suilleabhain, P.; Low, P. A.; Lennon, V. A. (January 1998). "Autonomic dysfunction in the Lambert-Eaton myasthenic syndrome: serologic and clinical correlates". Neurology. 50 (1): 88–93. ISSN 0028-3878. PMID 9443463. 
  48. ^ Mechem, C. C.; Walter, F. G. (June 1994). "Wound botulism". Veterinary and Human Toxicology. 36 (3): 233–237. ISSN 0145-6296. PMID 8066973. 
  49. ^ Taraschenko, Olga D.; Powers, Karen M. (June 2014). "Neurotoxin-induced paralysis: a case of tick paralysis in a 2-year-old child". Pediatric Neurology. 50 (6): 605–607. doi:10.1016/j.pediatrneurol.2014.01.041. ISSN 1873-5150. PMID 24679414. 
  50. ^ Witoonpanich, Rawiphan; Vichayanrat, Ekawat; Tantisiriwit, Kanit; Wongtanate, Manas; Sucharitchan, Niwatchai; Oranrigsupak, Petchdee; Chuesuwan, Aphinya; Nakarawat, Weeraworn; Tima, Ariya (March 2010). "Survival analysis for respiratory failure in patients with food-borne botulism". Clinical Toxicology (Philadelphia, Pa.). 48 (3): 177–183. doi:10.3109/15563651003596113. ISSN 1556-9519. PMID 20184431. 
  51. ^ Sandrock, C. E.; Murin, S. (August 2001). "Clinical predictors of respiratory failure and long-term outcome in black tar heroin-associated wound botulism". Chest. 120 (2): 562–566. ISSN 0012-3692. PMID 11502659. 
  52. ^ Wongtanate, Manas; Sucharitchan, Niwatchai; Tantisiriwit, Kanit; Oranrigsupak, Petchdee; Chuesuwan, Aphinya; Toykeaw, Sukumal; Suputtamongkol, Yupin (August 2007). "Signs and symptoms predictive of respiratory failure in patients with foodborne botulism in Thailand". The American Journal of Tropical Medicine and Hygiene. 77 (2): 386–389. ISSN 0002-9637. PMID 17690419. 
  53. ^ Canada, Health. "Botulism - Guide for Healthcare Professionals - Canada.ca". www.canada.ca. Retrieved 2017-10-08. 
  54. ^ Canada, Health. "Botulism - Guide for Healthcare Professionals - Canada.ca". www.canada.ca. Retrieved 2017-11-18. 
  55. ^ Varma, Jay K.; Katsitadze, Guram; Moiscrafishvili, Maia; Zardiashvili, Tamar; Chokheli, Maia; Tarkhashvili, Natalia; Jhorjholiani, Ekaterina; Chubinidze, Maia; Kukhalashvili, Teimuraz (2004-08-01). "Signs and symptoms predictive of death in patients with foodborne botulism--Republic of Georgia, 1980-2002". Clinical Infectious Diseases. 39 (3): 357–362. doi:10.1086/422318. ISSN 1537-6591. PMID 15307002. 

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