Table 1.

Comparative Diagnostic Methods for Clostridium botulinum. Compiled with data from: [3,60,61].

Diagnosis Method	Description	Advantages (A) or Disadvantages (D)
Mouse bioassay	Intraperitoneal inoculation with a suspicious sample in mouse 1, and neutralisation with polyvalent antitoxin or boiling in mouse 2. If (+), mouse 1 shows clinical signs (ruffled fur, posterior third paralysis, wasp waist, death, etc.). Mouse 2 shows no signs. If (−), both mice survive. Types the BoNT of the outbreak if a neutralisation test is done with specific antitoxin.	(A): Detection of toxin in serum, biological samples (stool, food and gastric contents), environmental samples (sediments) and crops. (A): High sensitivity and specificity. (D): Detection takes one to several days. (D): Ethical issues. (D): Difficult interpretation of results due to nonspecific signs in mice or prior death.
Cultivation and isolation	1º. Cultivation in liquid medium “chopped-meat-glucose-starch”. 2º. PCR confirmation of Clostridium Botulinum. 3º. If (+), solid medium culture, “blood agar”.	(D): Non-selective culture media. (D): Some strains lose the “bot” gene phage that encodes toxins, preventing their characterisation in mouse bioassays or PCR.
ELISA	It is carried out using polyclonal antibodies against a semi-purified toxic complex of BoNT.	(A): Analyses greater nº of samples than bioassay. (D): Less sensitive and specific than the bioassay. (D): False + due to the detection of inactive toxins that cross-react with other toxins. (D): False − due to the genetic variability of toxins.
Real-time PCR	1º. Sample enrichment in a culture broth to germinate the spores, increase the number of microorganisms and dilute inhibitors. 2º. Performance of PCR where millions of copies of specific target genes of the microorganism are produced.	(A): Sensitive and specific. Faster than culture and bioassay; allows analysis of more samples. (A): Enables ecological and epidemiological studies. (A): Detects BoNT C, D in environment and tissues. (D): Only reverse transcriptase PCR detects gene and toxin activity. (D): False + due to the detection of dead cells. (D): False − due to loss of the “bot” gene.
Mass spectrometry	In vitro detection of the peptides that form botulinum toxins after cleaving SNARE proteins at specific points.	(A): Detects biologically active toxin. (A): High sensitivity and specificity. (D): Only commercially available for type A toxins. (D): Expensive equipment & specialised personnel.