Botulism scare: how is Clostridium botulinum and its toxin detected?

By Siouxsie Wiles 05/08/2013 15


With news that all Nutricia Karicare Infant Formula Stage 1 and Stage 2 Follow-on formula should be avoided, the Fonterra botulism scare continues to rumble on. Fonterra Managing Director Gary Romano appeared on Campbell Live tonight where he said that it was the toxin rather than the bacterium Clostridium botulinum that had been identified in the whey powder. This is in contrast to everything that has been said so far and must be a mistake; it would make the scare a much bigger one than it is at the moment.

If it was the bacterium that was identified as the contaminant, it was most likely to be spores, produced by the bacterium as a way of surviving adverse conditions. The spores can hang around in the environment for years, until conditions are right for them to germinate and produce actively growing C. botulinum which can then produce the toxins that cause botulism*. As the conditions for germination and growth are very specific, the likelihood of spores germinating are actually relatively low, hence why botulism is extremely rare in developed countries. If the powder was contaminated with one of the actual toxins, a kilogram of which could kill the entire human population, then the chances of contracting botulism become much more real. Given that no cases have been reported to date, this scenario seems unlikely.

Fonterra have said that they have traced the contamination back to three batches of whey powder produced in May 2012 and questions are now being asked about why it took the company until the 31st July 2013 to inform the government and its customers of the contamination. Speaking on Campbell Live tonight, Gary Romano said that one particular standard test came back with a higher than usual result so they investigated, despite the result not actually being outside of the allowable limits. So what are the standard tests for C. botulinum? The US Food and Drug Administration (FDA) information on testing for C. botulinum toxin dates back to 2001, and states how to test for the bacterium itself and the toxin.

Testing for viable C. botulinum

C. botulinum comes in four ‘varieties’ known as groups I, II, III and IV. Most outbreaks of human botulism are caused by group I or II, while group III organisms mainly cause disease in animals. Group I and II strains can be differentiated on the basis of their ability to break down proteins and sugars. Group I organisms can break down proteins, while Group II organisms break down sugars instead. To complicate matters further, seven different types of toxin (A to G) have been identified, not all of which are harmful to humans.

In order to see if the bacteria are present, samples are used to inoculate an enrichment media which will contain all the nutrients C. botulinum needs to grown and which has had all the dissolved oxygen removed. The FDA suggests using cooked meat medium which is then incubated at 35°C. It’s worth noting here that the four different groups of C. botulinum have different optimum temperatures for growth**. After 5 days of incubation, the culture should be examined for turbidity, gas production, and digestion of meat particles. If there is any bacterial growth, some of the culture should then be Gram-stained and examined microscopically to look at the shape and size of the bacterial cells, and whether there is any evidence of spore formation. Apparently, a typical clostridial cell resembles a tennis racket! The FDA guidelines suggest testing the culture for the presence of toxin after it has been growing for 5-15 days.

It is worth remembering that as C. botulinum is so rarely found in dairy products, samples wouldn’t have been grown in this enrichment media as a first port of call. It’s likely they would have started more generically like for the Clostridium family, other species of which include the hospital superbug C. difficile, C. perfringens which can cause food poisoning and gas gangrene, and C. tetani which causes tetanus.

So to recap:
Step 1: Does the sample contain a species of Clostridia?
Step 2: Is it C. botulinum?
Step 3: Which group does it belong to?
Step 4: Does it produce a toxin, and if so, which type?

Whilst definitive, you can begin to see how it can take a little time to get a final confirmation that a sample contains C. botulinum, and the smaller the level of contamination, the harder it will be. No wonder then that the food industry is keen for more rapid, and sensitive diagnostic tests. A paper published in the Journal of Medical Microbiology in 2010 developed a real-time PCR assay that could simultaneously detect any of the A, B, E and F toxin genes in a given sample at a sensitivity of about 130–840 fg genomic DNA, which equates to about 50-330 genomes (1).

Testing for toxin

The gold standard for toxin testing seems to be a mouse bioassay. What this means is that suspected toxin samples are injected into the abdominal cavity of mice at different dilutions, and the animals monitored for signs of illness over a 48 hour period. If the toxin is present, mice injected with the sample solution will show signs of botulism. The time it takes for them to become unwell will depend on the concentration of toxin present in the sample injected. To find out which toxin is present, groups of mice are also injected with antitoxins to each of toxins A, B, E and F. If the correct antitoxin is injected alongside the sample, the toxin will be neutralised and those animals will not show any signs of botulism. Again this assay will take some time to perform.

While searching for diagnostic assays, I came across a paper in the journal Veterinary Record (2) in which the authors state that the numbers of cases of botulism in cows is increasing. They report finding toxin and C. botulinum from milk and udder samples. The article is not open-access so I’ve only been able to read the abstract and I’m not sure what types of C. botulinum they found but it may just be the types that are not harmful to humans.

References

1. Satterfield et al (2010). A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F. Journal of Medical Microbiology. 59: 55-64.

2. H. Böhnel, and F. Gessler (2013).Presence of Clostridium botulinum and botulinum toxin in milk and udder tissue of dairy cows with suspected botulism. Veterinary Record, 172:397 doi:10.1136/vr.100418

*The toxins prevent acetylcholine from being released from the motor nerve endings causing flaccid paralysis and symptoms of blurred vision, drooping eyelids, nausea, vomiting, diarrhea and/or constipation and cramps. In severe cases it leads to paralysis of the breathing muscles and causes respiratory failure.

**Group I = 35-40 degrees, Group II = 18-25 degrees, Group III = 40 degrees.


15 Responses to “Botulism scare: how is Clostridium botulinum and its toxin detected?”

  • How disturbing that a practising microbiologist affiliated to a NZ research and teaching institute can’t get quick and easy access to a current scientific paper that has direct relevance to our economy and health. I thought it was only folks like me in retirement land that had to put up with this nonsense.

  • Hi Souxsie;
    Thanks for a superb summary of the testing regimes. Oh that some of this informative stuff would enter the popular media so that some of the current hysteria could be tempered with rational reason.

    One thing I have been wondering is whether the “new test not generally used” that Fonterra referenced as the source of the first alarm might have been some form of genetic test. Thoughts? If so the propensity here for a false positive might be a factor to enter the fray down the track.

    On the matter of increasing botulism in cattle, I too have read that it is on the increase, in the USA at least. This is being attributed to increasing silage use and greater prevalence of badly-made silage (which of course is anaerobic) leading to higher risk of C botulinum affecting cattle. However I haven’t seen any reference to increased incidence in dairy products as a result, nor of human cases traced to dairy sources.

    Further, I understand that the C botulinum types prevalent in NZ are those that affect avian hosts (particularly waterfowl). When I was a micro student 40 years ago we were taught that there was NO human botulism strain known in NZ. So far as I am aware since then only two cases of human botulism have ever been reported in NZ and both were tracked to badly processed home canning of mussels and watercress where high vegetative growth had occurred, a far more predictable risk source than dairy products.

    • Hi MainlyMe! I’m going to put up a follow up post with some more resources I’ve found. One of them is a paper written by Graham Fletcher and colleagues at Plant and Food about the prevalence of C. botulinum in water samples from around NZ. Of 501 samples they tested, one came back positive for toxin A by PCR, which they thought had come into the water via a terrestrial source. Interestingly, none of the samples gave toxicity in the mouse bioassay.

  • As a retired microbiologist I suggest that the technical information to date from Fonterra is wooly. Nobody, including the Food Safety Authority have yet made it clear whether we are dealing with an organism or toxin – Fonterra use the word toxin. Comment to date makes me sceptical. Even if toxin is found it may not be a ‘toxic’ toxin. To my knowledge no mouse toxin/antitoxin testing has been reported. I am waiting to be contradicted. Scientificinformation has not come out clearly in this exercise.

    • Hi Alan

      Fonterra don’t seem to have released any scientific info! Gary Romano said toxin on Campbell Live but then said it was actually the organism when he was interviewed on Morning Report on Radio NZ. All the press releases from MPI have some organism. Give the time it has taken Fonterra to make the info public, I would have thought they could have told us the strain and potential toxin type, as well as the level of contamination at the very least.

  • Tks for info here.. plus links I’ll try get back to.

    From yr reading and given single toxin expression per strain is there evidence of multiple C.bot strains at any human flora site? An example may be the jejenum whose pH would seem to me suitable to C.bot?

  • Re the published result (Fletcher et al) that “one of 501 water samples tested positive for Toxin A”, we should also remind ourselves that this same toxin is exactly the Botox of cosmetic commerce. Just wondering whether the very recent influx of Botox into commercial chains has any consequences in environmental appearance of this toxin independent of the bug. Hmmmm……

  • […] Botulism scare: how is Clostridium botulinum and its toxin detected? | Infectious Thoughts In order to see if the bacteria are present, samples are used to inoculate an enrichment media which will contain all the nutrients C. botulinum needs to grown and which has had all the dissolved oxygen removed… […]

  • […] Botulism scare: how is Clostridium botulinum and its toxin detected .With news that all Nutricia Karicare Infant Formula Stage 1 and Stage 2 Follow-on formula should be avoided, the Fonterra botulism scare continues to rumble on. […]