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bbd
Tom Parsons from NBC
has interest in animal wellbeing, 
and measuring stress in animals
today will discuss pathogenesis of 
clostridial diseases

Q: briefly describe common aspects of botulism and 
tetanus pathophysiology
if the toxins create similar molecular lesions, 
why are clinical presentations so different?

we understand molecular 
pathology of clostridial diseases very well. 
botulism and tetanus have 
strikingly different clinical signs, but identical molecular actions of 
the toxins.

the molecular pathogenesis of Botulism and Tetanus are the 
same. the clinical signs are extremely different - that's the point he's 
making repeatedly today.

Molecular pathology of clostridial disease:


-clinical aspects of tetanus and botulism
-physiology of synaptic 
transmission
-pathogenesis of clostridial disease
-final comments


Questions with which to terrorize students during rounds:
-what is the 
molecular action of clostridial toxins?*
-why if the action is so similar 
are the signs so different?*
-why are there no food-borne outbreaks of 
tetanus?
-what drugs are contraindicated in botulism, and why?
-what new 
therapeutic options are available?
-why is a horse so sensitive to 
botulism poisoning?

Clinical aspects of tetanus and botulism:

Tetanus 
or botulism:
PE
epidemiology
rule-outs
therapeutic plan

slide show:


he's showing some slides - ID the disease, then the clinical signs of it. 
In two words, describe clinical presentation of botulism - flaccid 
paralysis. Tetanus? spastic paralysis.

rigid foal - tetanus. legs and 
neck extended, neck arched, sawhorse stance
floppy foal - hay falling out 
of mouth - dysphagia, not standing up
shaker foal  - botulism - muscle 
tremors, toe scuffing, flaccid paralysis
rigid foal - cotton in ears to 
reduce stimulation, sawhorse stance
(remember with tetanus there is 
generalized disinhibition of neural responses so they overrespond to 
stimuli)
floppy calf - botulism, tongue hanging out
rigid piglet - 
tetanus - rare in pigs
botulism - loss of tail tone 
tetanus - cow with 
sawcow stance :)
cows falling down - botulism - falling down - ataxic - 
"herd ataxia" botulism is one of the few things that will cause a herd 
outbreak of herd ataxia.

tetanus - flick of the haw - prolapsed third 
eyelid; erect ears.
botulism - down cow; horse with tongue hanging out; 
decreased tongue tone is most common presenting complaint in horses with 
botulism b/c toxin is ingested, spreads locally, these muscles are first 
to be affected.

lamb with tetanus - not that uncommon

Botulism:
PE: 
symmetrical flaccid paralysis of skeletal muscle w/o loss of sensation

foal: "shaker foal" - muscle weakness and tremors, gait abnormalities - 
minimal limb flexion, toe scuffing; reduced tongue and tail tone.
adult 
horse: more variable - muscle weakness, hypersalivation, colic, racing 
poorly, dysphagia
cow: downer cow, atypical milk fever, multiple animals 
in herd, non-peri-parturient cows, dysphagia, bradycardia.

epidemiology 
of botulism: clostridium botulinum is agent
gram positive, spore forming 
anaerobic rod; types A, B, Cz, Cb, D,E, F, G
routes of infection: 

ingestion of preformed toxin --cattle, horse
toxicoinfectious botulism -- 
foal (ingestion of organism, spores germinate, make toxin) -- in foal, 
b/c early in life mammalian stomachs are porous to protein.
wound 
botulism - rare, but can occur. wound gets infected and toxin is formed


therapeutic plan for botulism:
neutralize unbound toxin with antitoxin 
($$)
wait
nursing support, stall rest, AVOID aminoglycosides because they 
antagonize entry of calcium into nerve cells, and that will further 
compromize the neuromuscular junction function. 
vaccinate to prevent 


Tetanus:
general PE: stiffness, muscular rigidity, spastic paralysis, 
lockjaw
horse: flick of the haw, stiff ears, sawhorse stance
cow: 
stiffness, free gas bloat (cow can't pass gas as it should)

epidemiology 
of tetanus - clostridium tetani
another gram pos spore forming anaerobic 
rod
common in feces and soil
enters through puncture wounds, uterine 
prolapsees, other surgical sites like castration, dehorning, docking of 
tail. 

therapeutic plan for tetanus: clean wound, destroy organism, 
neutralize unbound toxin with antitoxin; control muscle spams with 
relaxants or sedatives; nursing support, vaccination.

so that's the 
clinical presentation.
botulism -flaccid paralysis
tetanus - spastic 
paresis

go back in time now and think about synaptic physiology

basics: 
three seminal ideas here - vesicle hypothesis, calcium hypothesis, snare 
hypothesis.

considering the stellate ganglion of the squid -- you see 
presynaptic element and postsynaptic element. electrical impulse comes to 
end of one nerve, chemical impulse travels through space, elicits 
postsynaptic electrical response.

there is a bit of a delay - about 200 
microseconds. that's pretty rapid. people always assumed it couldn't be 
chemical b/c it is so fast, but it is indeed chemical. but we digress. 


the underlying cause of these clostridial diseases is indeed a 
biochemical lesion as you will find out soon.

essentially your muscle 
fiber is there with nerve fiber coming onto it with several terminal 
boutons - aggregates of synaptic vesicles are lining up at presynaptic 
membranes, releasing stuff, which binds to postsynaptic receptors on 
muscle cell, leading to activation of cell.

"sparks" vs "soup" debate - 
electricity vs chemistry
chemical synapse predominate
structural 
specialisations: synaptic boutons - release sites; synaptic clefts - 
extremely restricted space through which transmitter diffuses. this is a 
very small site, allowing very rapid diffusion.

vesicle hypothesis:

packets of NT:  spontaneous release of NT - if you sit there and monitor 
a muscle cell without stimulation, you see spontaneous activity occuring. 

when transmission is evoked, there is quantal release. spontaneous events 
had a characteristic unit size, ok? then, evoked responses were much 
bigger, in these sizes that were multiples of the spontaneous size. so 
the idea is, somehow NT is released in packets of uniform size. you can 
have one packet released, or 2 or 3 or 10.

when the EM was invented they 
got pictures showing these little bubbles (vesicles) aggregating at 
preynaptic membrane...little intracellular vesicles, subcellular 
organelles -- the hypothesis they came up with then waas that these were 
the packets of NT which is pretty insightful for 35 yrs ago, especially 
since they turned out to be correct. so how does the NT get into the 
extracellular space/ The vesicles fuse with presynaptic membrane.  the 
morphological criterion for chemical synapse, then, was presence of these 
vesicles. and it was shown that these vesicles fusing with membrane 
correlated with release of NT. what regulates vesicle fusion? good 
question!

Calcium hypothesis - vesicle endocytosis triggered by Ca++ 
influx. the AP opens voltage gated calcium channels (specialized proteins 
in the presynaptic membranes), calcium enters the cell. this has 
exquisite temporal control, very very rapid. 

key points - synaptic 
vesicle approaches membrane, undergoes docking, fusion, exocytosis, 
(fusion/exocytosis occur after calcium influx), then endocytosis of 
vesicle and recycling.

ok, to get the rest of info we need, look at 
yeast. there is a process you may have heard of- transgolgi transport, 
remember that? things go across golgi, getting modified, and then get 
secreted. people studied this b/c they found this was a fusion mechanism, 
where vesicles bud off the RER and then glom onto and fuse with golgi, 
then bud off the other side, and so forth. they wanted to understand 
molecular biology of this and came up with the Snare hypothesis.

SNARE: 
intracellular transport is mediated by budding and fusion of vesicles. 
how is direction controlled? vesicleSNARE and targetSNARE proteins - 
determine specificity and direction of transport. SNARE = SNap REceptor. 
anyway these proteins would be on the vesicle membrane - vSNAREs - and 
would line up with the tSNARE of interest. this provided directionality. 
now, when they looked for these molecules in cells other than yeast, they 
found homology with proteins in synaptic vesicles. we knew there were 
many proteins on the synaptic vesicles but didn't know what they were; 
now we thought, Aha! maybe we know what is going on.

so point of that 
is, as it turns out, the v and t SNARES are substrate for clostridial 
toxin action.

in the nervous system we found putative SNAREs - tSNARE in 
presynaptic membranes were syntaxin and SNAP25; and vSNAREs on vesicles 
were VAMP/synaptobrevin. so they suggested that there was interaction b/w 
these proteins, and proposed a model for docking, activation, and fusion 
- the details of which were wrong but general principles were right.

so 
your VAMP comes down and docks, and is held tto syntaxin and snap25 until 
calcium comes in. 

pathogenesis of clostridial disease:

the toxins do 
stuff - they are holotoxins, first of all. they have characteristic 
structure - made as single chain polypeptides of about 150 kDa. tetanus 
makes one toxin, botulism makes 7 different ones.

how do they work? held 
together by disulfide chain - light chain L, heavy chain H, held by 
disulfide bond. the bond is cleaved by cellular proteases, making active 
form of toxin. 

diff b/w toxins - botulism but not tetanus is complexed 
with an additional, nontoxic protein, to protect from GI degradation. 
this is why ingesting tetanus toxin doesn't cause disease - it isn't 
protected from GI degradation by this extra protecting protein. 

both 
toxins attack motor neurons specifically and are taken in by endocytosis.


the H chain binds nerve cell receptor, starts endocytosis. toxin gets 
into lysosome, bond is broken, L chain is enzyme - starts clipping off 
SNARE proteins and blocking normal exocytosis of vesicles. 

Botox - 
remains in peripheral nerve terminal, has action there
TeTox - moves 
around - retrogradely transported to spinal cord in vesicles. goes 
through transcytosis, seletively binds inhibitory interneurons and acts 
on them - so that is why there is spastic paralysis - lack of function of 
inhibitory neurons. 

H chain - neuroselective binding region with high 
affinity
L chain - active part, blocks NT release

looking at the toxins 
- high degree of homology - most conserved region is L chain. we see in 
almost all these toxins that they have a repeating His-Glu-x-x-His motif. 
this is a zincbinding motif seen in metallo-endopeptidase proteins. this 
suggests the L chain functions as a Zn dependent protease.

possible 
therapeutic options: metallo-peptidase inhibitors: captopril (angiotensin 
converting enzyme), EndothelinCE ( phosphoramideon), Neural 
endopeptidase-24.11 (thiorphan)

final point - synaptic vesicle proteins 
- different toxins cut them in different places, so some cleave one 
protein, others cleave the other protein...tetanus toxin cleaves 
synaptobrevin, botoxes cleave various...

8 toxins, each cleaves a SVAP, 
homologous to the SNARE proteins in yeat. each toxin cleaves only one, 
and at a distinct, unique site.


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