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...Salmonella infection arising from contaminated food continues to be an immense problem with millions of cases occurring annually

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Salmonella Serology and Antisera Production...Salmonella infection arising from contaminated food continues to be an immense problem with millions of cases occurring ... – PowerPoint PPT presentation

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Title: ...Salmonella infection arising from contaminated food continues to be an immense problem with millions of cases occurring annually


1
Salmonella Serology and Antisera Production
...Salmonella infection arising from contaminated
food continues to be an immense problem with
millions of cases occurring annually throughout
the world. In addition to the misery caused,
financial loss is enormous. - WHO Global
SALM-SURV Laboratory Protocols, September 2002
2
- started working for Enterics in February, 2000
as part of the second year Chemical and
Biosciences Technology course at Red River
Community college - wrote 4 boards, and finally
won an indeterminate position - also work with
Listeria sp., doing serology and antisera
production
3
The Salmonella serotyping system is probably the
best phenotypic bacterial typing system ever
developed. It has high discriminatory power and
provides information that has great
epidemiological significance.
Molecular typing methods such as PFGE can yield
supplementary information, but are so far not a
substitute for serotyping.... - WHO Global
SALM-SURV Laboratory Protocols, September 2002
4
Serotyping is a definitive typing method used for
epidemiological characterisation of bacteria.
Serotyping of Salmonella strains is carried out
by identification of surface antigens (LPS,
O-Antigen) and flagella antigens (proteins,
H-Antigens). Most commonly strains of
Salmonella express two phases of the H antigens,
but aphasic, diphasic, triphasic, and
quadriphasic variants are known. The definition
of serotypes is based on the antigen combination
present and is given in the "Kauffmann-White
Scheme" (Popoff and LeMinor, WHO Centre for
Salmonella Reference and Research on Salmonella,
Institut Pasteur). - WHO Global SALM-SURV
Laboratory Protocols, September 2002
5
O Antigens - heat stable - somatic - composed of
phospholipid-polysaccharide complexes - granular
agglutination - subject to smooth (S) and rough
(R) variation - some are subject to lysogenic
conversion by bacteriophages
6
H Antigens - heat labile - occur in the
flagella - composed of flagellins
(proteins) - agglutination is rapid, loose, and
floccular - reversible phase variation occurs
7
Agglutination - Antibody-Antigen
interaction - antibodies in the specific antisera
agglutinate with the bacteria when the
corresponding antigens are present - seen as
particulate matter, or lumps forming - graded by
strength as 1, 2, 3, and 4 - 4 is a clear
background with 100 agglutination - O
agglutination is granular - H agglutination is
loose and floccular
8
Kauffmann-White Scheme - developed based on the
work of White (1925, 1926) and Kauffmann (1930,
1941, 1966) - made rapid, accurate serological
identification possible - White classified
serotypes by H antigens - Kauffmann grouped
serotypes according to O antigens - 2500
serotypes of Salmonella currently known, with
more added to the scheme every year - Kauffmann-Wh
ite scheme is updated every 5 years
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submitted sample
MAC plate
11
Bios
MAC plate
NA plate
submitted sample
semi-solid plate
12
Biochemical Identification - used to determine
Salmonella spp. - most Salmonella received are
ssp I - A short set of bios are done. - if
results are not typical of ssp I, then an
additional set of biochemicals (called
Kauffmann-White bios) are set up.
13
  • 90 or more positive reactions
  • 90 or more negative reactions
  • d different reactions given by different
    serovars

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typical Salmonella ssp I bios
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typical S. Typhi bios
18
Serotyping O Antigens - tested by slide
agglutination - autoagglutination indicates a
rough culture, which is subcultured further to
recover its smooth state - strains are first
tested in O sera pools (aka Polys) - depending on
which Poly O serum agglutinates, the individual
factors of that poly are then tested
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Serotyping H antigens - H antigen is flagellar,
so strains are plated onto a semi-solid media to
encourage motility - tested by slide
agglutination, then confirmed by tube
agglutination (which is more sensitive) - autoagg
lutination indicates a rough culture, which is
subcultured further to recover its smooth
state - strains are first tested with H sera
pools (aka Polys)
24
- depending on which poly H serum agglutinates,
the individual factors of that poly are then
tested - individual H factors are then confirmed
by tube agglutination - phase inversion is then
attempted
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Phase Inversion/Suppression - a few drops of
antiserum corresponding to the H factor
identified are placed into an empty
plate - melted semi-solid agar is then poured
into the plate and slightly agitated to
distribute the serum evenly throughout the
media. - once the "suppression" plate has
solidifed, the strain is plated out - after
being incubated overnight, H serology is again
performed, testing first for the H factor
previously identified - if the strain is motile,
and the previously identified H factor is
absent, another phase may be present and can be
identifed using the H serology methods
previously described.
29
Motile
Non-motile
30
Antisera Production - 99 of all antisera used in
serology is produced in house - WHO publishes
guidelines on antisera production which cover
what strains to use for immunization, and which
strains are recommended for the absorption of
cross reacting factors - the strain used for
immunization is selected and grown on solid (for
O) or semi-solid (for H) media and then
harvested. - strains for the production of H
antisera must be in the proper phase
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- O antigens are boiled, H antigens are
formalized - antigens are centrifuged and washed
several times - antigens for immunization
(vaccines) are standardized using a
spectrophotometer and a mathematical
formula - standardized antigens are then sent to
Animal Services for immunization
34
- SPF New Zealand White rabbits are used - the
inoculation schedule and the amount of
inoculation injected vary depending on whether
an O or H antiserum is being produced - a trial
bleed is taken at the end of the inoculation
schedule and sent back to be titred - if the
titre is acceptable, the rabbit is bled out by
cardiac puncture - Animal Services separates the
serum and sends it back - 1 rabbit can produce
as little as 30mL of serum, or as much as 80mL
of serum.
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Testing/Establishing Titre of Antisera - once
"crude" antiserum is received from Animal
Services, it is filtered and merthiolate is
added - crude sera are tested on slide using
serial dilution, using positive and negative
(cross reacting factors) controls. - if some or
all of the negative controls are positive at the
same titre as the positive controls, they must
be "absorbed" out - absorption is accomplished
by plating, harvesting, centrifuging, and
washing the cross reacting antigens - a portion
of the crude serum is added to the antigen cells,
mixed well, and incubated
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- the serum and antigens are centrifuged, the
serum is drawn off, and retested with the same
positive and negative controls on slide. - this
serum is now considered an "absorbed serum", once
testing is complete, it is filtered and
stored - several absorptions may be necessary to
remove all cross reacting factors - titre is
established by testing all controls with a serial
dilution - titre is set at the highest dilution
that will give a 3 agglutination with all
positive controls used - once a serum no longer
cross reacts with any cross reacting factors it
can then be tested in tube (again using a serial
dilution) - since the tube method is more
sensitive than the slide method, more
absorptions may be necessary
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- after every absorption the serum must be tested
on slide since the absorption usually causes the
titre to drop - once serum is titred it can be
diluted and used in slide or tube - multiple
absorptions can result in no useable titre, in
which case the serum must be discarded and the
whole process must be repeated - O antisera have
much lower titres than H antisera
41
QC - although each serum is tested extensively
for cross reacting factors when it is titres, QC
still has to be performed on all diluted
antisera in use - this twice yearly QC only uses
selected positive controls and ensures that the
antisera in use is still working
properly - internal and external proficiencies
are done on a regular basis - proficiency panels
are also prepared by us and sent to other labs
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Miscellaneous Facts - Salmonella has been
associated with outbreaks caused by chocolate,
pepper, canteloupes, alfalfa sprouts, pig's ears
(dog treats), almonds, lettuce, and keeping
reptiles as pets - most reptiles carry and shed
Salmonella in their feces (natural flora) - CDC
estimates that 74,000 Salmonella cases caused by
exposure to reptiles are reported each year in
the US - our lab will be going for accreditation
by ISO 17025 March, 2005 - currently working on
a microtitre system to replace slide and tube
testing for H factors
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