Case Study 7 Botulism

Pages 9, 10, 11, 21, 33, 34, 35 are not questions from the case study Questions of the case study are marked by this symbol at the top of the slide

Presentation

BOTULISM

Case study 7 : Clostridium botulinum

INDEX

1. Findings on field

8. Prevention

2. Clostridia

9. Treatment

3. Def. diagnosis

10. Vaccination

4. Neurotoxins

11. Medical uses

5. Infection

12. Case study

6. Signs and symptoms

13. Team

6. Mechanism

14. Thanks

Introduction to Clostridia

From the field to the clinic

Clostridia

· Gram +· Anaerobes · Spore producing · Multiple species are pathogenic

Wirtz-Conklin staining

Methods for diagnosis

Diagnosis is confirmed by finding BoNT in patient's serum or faeces. Finding the bacterium in GIT samples, wound swabs and faeces is not sufficient except in infants.

Mouse bioassays including mouse lethality and mouse neutralization tests

Molecular assays including anaerobic cultivation of Clostridia and PCR tests

In vitro assays including ELISA and endopeptidase assays

Describe the method of toxin-neutralising test on mice. Which toxic substance is diagnosed this way ?

Mouse lethality test(more used)

Mouse neutralization test

1. Intraperitoneal injection of sample into laboratory mice.

1. Intraperitoneal injection of sample into laboratory mice.

2. We also inject a control group with physiological solution and a third group with cooked sample (to destroy the BoNT)

2. Intraperitoneal injection of antitoxins into laboratory mice.

VS

3. The mice that received correct type of antitoxins survive.

3. Fuzzy hair, muscle weakness and respiratory failure.

4. Detection of type of botulotoxin (A, B, C, D, E or F)

4. The mice showing signs of botulism received a sample positive for C. botulinum

Toxin antibody

Anti-antitoxin

Detection by sandwich ELISA

· Harder to find high quality antibodies than lab mice

· All toxins are detected even if they are inactive = false-positive

· Genetic variation may result in decreased affinity to Ab = false-negative

· Food and fecal components from samples decrease sensitivity

Molecular technique : PCR

· Multiplex PCR enables the simultaneous detection of the botA, botB, botE botF genes.

· C. botulinum strains carrying silent toxin genes may cause false-positive results.

· Target the neurotoxin genes but they do not detect active neurotoxin.

· Point mutations at the site of PCR primers may cause false-negative results.

· PCR doesn't detect spores, PCR, enrichment is required to germinate spores first.

Cultivation of C. botulinum

C. botulinum requires strict anaerobic conditions for growth.All culture media, glassware, pipettes, tubes must be deoxygenated every time we work with C. botulinum. Constant anaerobic environment must be maintained.

Botulism detection in France

Detection in humans

Detection in animals

1. Based on the detection of BoNT in serum and stool

1. Before 2010 : detection of BoNT in serum using the mouse bioassay

2. Or detection of the neurotoxigenic C. botulinum in stool or gastric contents

2. But, does not discriminate between mosaic and non-mosaic forms.

VS

3. The bacterium and its toxin can also be tested in suspected food

3. Today, detection of C. botulinum in faeces, digestive contents as well as organs

4. Gold standard for the diagnosis of botulism is the mouse bioassay

4. Method = PCR after enrichment step in anaerobic broth.

Botulism in different animal species

Types of toxins and sources of infection

Name the types of botulotoxin and say which animal species are influenced by those types. Which animal species is most sensitive to botulotoxin?

Botulinum neurotoxins (BoNT)

· A, B, E, F in humans · B, C, D in cattle · B, C, (A) in horses (most sensitive species) · C, D in birds · E in fish Dual-toxin-producing strains Strains producing only one type of toxin but carrying a silent gene for another

Also reported :

What are the sources of botulotoxin for different animal species ?

Ways of infection

Through wounds

Alimentary

Toxo-infectious

Once the wound has closed, there is an anaerobic environment, perfect for the development of C. botulinum. This infection can be observed in intravenous drug abusers.

Ingestion of preformed BoNT. The case-fatality rate of food-borne botulism in developed countries is 5 to 10%.Worldwide, more than half of the cases (52%) were due to type B toxin, with types A and E accounting for 34% and 12% in 1995.

Ingestion of C. botulinum spores that germinate in the GIT and start producing BoNT. Babies and people with altered intestinal flora due to abdominal surgery, prolonged antimicrobial treatment are particularly vulnerable.

Risk for infant

What are the sources of botulotoxin for different animal species ?

Infection in animals :

Pets (dogs and cats) can be infected by the same ways as humans : incorrect storage meat, repetitive defrosting, improper can preservation. Also by eating cadavers. Horses might come accross the toxins in contaminated hay (from soil = BoNT-B, from cadavers = BoNT-C). This happens mainly haylage bales in plastic due to the anaerobic environment.

Describe zoohygienic measures in botulotoxin poisoning for those different animals species.

CarnivoresCold chain maintenance for meat products : every time the meat is defrosted the Clostridia can start producing some toxins. Extra precautions for homemade canned products (not only meat, some BoNT were found in canned vegetables after soil contamination). Horses Removing the cadavers from pasture, making sure there is no dead rodents or birds in the haylage bale that would allow Clostridia to grow.

Describe clinical signs for the different animal species.

Clinical signs of botulism (incubation = 18-36h)

· First of : apathy, fatigue = non-specific signs

· Difficulty standing and walking, difficulty keeping the head up

· Cranial muscle paralysis (dilated pupils, tongue out, lacrimation because no blinking, difficulty swallowing and speaking)

What are the probable sources of Clostridium bacteria in this case ? What preventive measures would you recommend in this case ?

Intoxication of waterfowl

As they are anaerobic, clostridia thrive in sediment of eutrophicated ponds. Ducks might ingest them with their normal food or in invertebrates. Birds are natural hosts of Clostridia. They can be found in healthy bird's GIT with no consequences but when the bird dies, they will replicate and be eaten by the maggots that will subsquently be eaten by another bird and disrupt the microfloral balance.

Prevention

What is the mechanism of action of botulotoxin?

Acetylcholine (ACh)

VAMP / Synaptobrevin

BoNT mechanism

Botulinum neurotoxins are proteins with zinc-endopeptidase activity. The light chain is responsible for cleaving the proteins involved in ACh vesicle fusion and release from the presynaptic neurons. BoNT inhibits transmission of impulse through the neurons.

Syntaxin

SNAP-25

Another diagnostic method developing :

Endopeptidase assays

Based on specific cleavage of synaptic proteins by different botulinum neurotoxins. Then immunological, fluorescent or mass spectropmetry detection of the cleaved peptide.

Prevention & Treatment

Are there any vaccines available ?

Heating

The neurotoxins are heat-labile and will be destroyed within minutes at temperatures above 80°C. Not the spores though.

Acidifying

Most Clostridia cannot develop in pH inferior to 4 or superior to 9. Acidifying cn be combined with freezing or salting to prevent the growth of the bacteria and the production of toxin. Yet, the toxin does survive to stomach pH

Freezing

Freezing will prevent further growth of the bacteria and production of toxins. However, the spores and the toxins will not be destroyed.

What is the therapy in botulotoxin poisoning ?

Treating Botulism

Very ealry after poisoning : gastric lavage or emetics only if the animal is not paralyzed yet (risk of aspiration pneumonia from vomit). Activated charcoal is also possible if the toxin is sitll in GIT.

Early, before the toxin enters the nervous system, we can give injection of antitoxin (immunoglobulins). But it is expensive and there is a risk of anaphylactic reaction.

Administration of physostigmine to inhibit AChE and allow more ACh in the synapses. Too early will cause similar effect as carbamates and OP poisoning, too late will have no effect.

Impossibility swallowing -> give food parenterally

Is it possible to do the vaccination of animals/humans ?

Vaccines in humans

In the USA, the scientists developped a ‘‘formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine’’ more simply called ‘‘Pentavalent Botulinum Toxoid Vaccine’’. It was an experimental vaccine for 5 toxins but throughout the years it lost potency and was eventually discontinued in 2017. In France, there is also a toxoid vaccine used, not the same and not available to the general public. This vaccine is used only by people at risk such as those working in laboratories or soldiers threatened by the use of biological weapons.

Is it possible to do the vaccination of animals/humans ?

Animals

Vaccination is indicated as soon as there is clinical suspicion of botulism in the herd. In France, the approved vaccine is ULTRAVAC BOTULINIUM for type C and D BoNT. It is used for both sheep and cattle There is a vaccine for poultry as well. It protects from type C. However, preventive vaccination of an entire poultry farm would be too expensive.

Is it possible to do the vaccination of animals/humans ?

In endemic countries

It is way more common to see vaccinated animals in endemic areas than in Europe. For example, in South Africa, Botuvax is used on cattle, small ruminants and sheep. It protects against C and D toxins

Medical uses of BoNT

Injection of the toxin on purpose

Is botulotoxin intentionally used for some purposes ?

Laminitis in horses

Deep digital flexor

Inflammation of the lamina causing hoof wall detachment from the coffin bone, potentially leading to severe conditions due to stress from the animal's weight and movement. This stress is exerted by the deep digital flexor. BoNT-A (Botox) IM injection = reduction in DDF muscle activity.

Lamina

Coffin bone

Stringhalt = Recurrent hyperflexion of the tarsus potentially due to over-reacting upper motor neurons.

Is botulotoxin intentionally used for some purposes ?

BoNT-A (Botox) injections in the extensor digitorum longus, lateralis and lateral vastus muscles. There was only partial success obtained with this trial (the spasticity was not completely abolished).

Is botulotoxin intentionally used for some purposes ?

In a horse with lameness due to acute synovitis, the injection of BoNT-A into the affected joint results in reduced lameness. This demonstrates BoNT-A has pain-relieving properties.

BoNT as analgesic :

BoNT-A (Dysport) was also found effective as an adjuvant analgesic agent in post-operative bilateral radical canine mastectomy, reducing sensitivity by inhibiting neuropeptide release at nociceptive nerve endings.

The injection of BoNT-A (Botox) into the joints in dogs suffering from osteoarthritis showed significantly reduced pain for a few months.

Is botulotoxin intentionally used for some purposes ?

Medical uses in dogs

Prostate

Esophagus

Stomach

It is very common for dogs to have benign prostatic hyperplasia. Injecting BoNT-A (Botox or Dysport) into each prostate lobe showed reduced size and firmness, increased glandular atrophy and apoptosis, without complications, side effects, or semen quality impact.

Functional obstruction of the lower esophageal sphincter (due to the loss of inhibitory myenteric neurons) leading to a lack of relaxation after swallowing. BoNT-A (Botox) injections around the esophagogastric junction.

Delayed gastric emptying in dogs was effectively treated by Botox injections into the pylorus, to relax the sphincter and allow the drainage of stomachcontent.

Real life Case Study

Namdong's reservoir, South Korea

Namdong, Incheon (SK)

In October 2008, 2000 birds died.More recently, in 2016, there was another outbreak.

Namdong 2016

Opening of Namdong's floodgates (adjacent to the Yellow Sea) to decrease the water temperature and the nutrient load (N, P). Additional removal of carcasses.August 17 to 22, 2016

Collection of cecal content, soil samples and maggots

Two paralyzed and four dead spot-billed ducks (Anas poecilorhyncha) July 15, 2016

Over the next two months, 697 wild birds were found paralyzed or dead.

All maggots and soil samples are positive for type C by mouse bioassay

References

1. The Journal of Veterinary Medical Science

8. ANSES

2. Poultry DVM

9. Medical uses

3. Botulism Vaccine in South Africa

10. Botulism Vaccines

4. Laboratory Diagnostics of Botulism

11. Botulism in France WHO

5. Botulism Surveillance in France

12. Botulism WHO

6. Vaccine for Ruminants in France

13. Botulism in France AFSSA

Preventive measures

Duck pond is already contaminated

Move the ducks to an other pond or a different holding place.It is not good to give paralyzed ducks an access to a pond, as they cannot hold their head up, they might drown. Make sure to remove the dead bodies as soon as possible to prevent further contamination. Start treatment / supportive care of the live ducks. Treat the pond. Clostridia thrive in warm and anaerobic water. Make sure to get oxygen inside the pond and decrease the temperature. Don't forget to deal with the maggots and invertebrates. They are not affected by the BoNT and might still contain high amounts in their tissues. Don't put the ducks back to the pond if they are just going to eat them and get infected again.

Risk for infants

Toxo-infections

For infant botulism, in addition to group I C. botulinum types A and B, Bf, cases due to type E and F toxins have been reported. Infant botulism typically affects babies under 1 year of age, with the youngest reported patient being only 54 h old Honey is a typical product not recommended for infant younger than 1 year old as it potentially contains Clostridium's spores.

https://pubmed.ncbi.nlm.nih.gov/15730901/

Inhalation

Not common at all

Inhalation botulism may result from aerosolization of the neurotoxin. It is rare but can happen since the neurotoxin can enter the body through mucous membranes of the respiratory tract (also the eyes). These cases are not often reported but they could be a finding in bioterrorism.

Human and animal botulism surveillance in France from 2008 to 2019

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9730534/

Horse vaccine

In USA

Due to high number of cases in the West coast of the USA, an endemic vaccines is used for horses. It protects them from BoNT-B which is usually transmitted through soil contamination (contrary to BoNT-C transmitted by cadavers as it is physiologically present in the GIT of some animal species, for example birds).

Hyperexcitation

Treatments for dogs

BoNT-A (Botox) has a beneficial impact on dog myoclonus (= involuntary muscle twitching). By targeting affected muscles with toxin injections, walking and motor functions were improved without significant adverse effects. Local subcutaneous injections of BoNT-A (Dysport) were also effective in the treatment of primary dog blepharospasm (= abnormal, uncontrolled contraction of the eyelid muscles).

Light Chain

The light chain, is a zinc protease responsible for cleaving the SNARE proteins involved in acetylcholine vesicle docking and fusion to the presynaptic membrane. Inhibition of the neurotransmitter release causes paralysis of the muscles and eventually death due to paralysis of the respiratory muscles and subsequent suffocation. Once inside a neuron, BoNT-L can persist in an active form for up to several months, depending on the BoNT serotype.

Heavy chain

The heavy chain is responsible for the binding and translocation of the toxin across the synaptic membrane through specific receptors. Heavy chains has an N-terminal Translocation domain (HN) and a C-terminal Receptor Binding domain (HC). BoNT neurotoxicity is only possible if the heavy chain is present to bind and be uptaken by the neuron-specific receptors. Therefore, even if the heavy chain is not damaging the neuron by itself, it is necessary to induce the botulism signs.

Recent case in France

On 14 September 2023 WHO was notified a total of 15 cases of botulism including one death and ten hospitalisation into intensive care unit. The source of infection is the consumption of homemade preserved sardines served in a restaurant in Bordeaux during the week of 4–10 September 2023. Unfortunately, the restaurant attracting international visitors because of the Rugby World Cup. Local investigations have identified an estimated 25 persons through credit card receipts who have been exposed. Botulinum toxin B was confirmed in serum samples and in a food sample (sardines) on 14 September 2023.

https://www.who.int/emergencies/disease-outbreak-news/item/2023-DON489