VIBRIO CHOLERAE ANTISERA
Liquid stable antisera for the determination of O antigens of Vibrio cholerae
by slide or tube agglutination.
Introduction
Principle of the Test
V. cholerae are Gram negative aerobic or facultative
anaerobic comma shaped rods. They are motile
possessing a single polar flagellum.
Taxonomically V.cholerae is a homogeneous species
comprising organisms that are similar to each other
biochemically, share a common H (flagellar) antigen
and are closely related genetically.
Organisms identified as V.cholerae by their
morphological and biochemical features are taken
from plates (or broth cultures), emulsified in saline
and mixed with an equal volume of VIBRIO
CHOLERAE ANTISERUM. After a defined length of
time they are examined for antigen-antibody
interaction. A positive interaction is observable as
macroscopic agglutination and indicates that the
V.cholerae organism contains one or more antigen
specified by the antiserum. A negative interaction is
observable as a lack of agglutination and indicates
that the organism contains none of the antigens
specified by the antiserum.
The serology of V.cholerae is based on the Somatic
O antigen scheme of Sakazaki et al.1,2 Now more
than 80 serogroups have been identified within the
species.3 Serogroup O1 organisms comprise the
causal agents of most epidemic and pandemic
cholera outbreaks. Cholera is a highly contagious
disease characterised by severe diarrhoea (‘ricewater’ stools) due to toxin production by the
organism.
In the early 1990’s a new serotype of V.cholerae was
found to be the causative agent of a pandemic in
India and Bangladesh, and was named serotype
O139. It gives rise to an infection as serious as those
caused by serotype O1.4,5
Strains of V.cholerae serotype O1 may be subdivided
with absorbed antisera into variants or subtypes
called Ogawa, Inaba and Hikojima.2 These variants
share three somatic antigens - a, b and c. Absorption
of O1 antisera with Ogawa organisms produces a
serum which agglutinates Inaba and Hikojima strains.
Similarly, absorption of O1 antisera with Inaba
organisms produces a serum which agglutinates
Ogawa and Hikojima strains. Unabsorbed serum
(containing a, b and c) called ‘polyclonal V.cholerae
antiserum’ agglutinates all three O1 variants.
Description and Intended use
VIBRIO CHOLERAE ANTISERA are a set of antisera
for the agglutination of specific V.cholerae O1
antigens and the O139 (Bengal) antigen.
Packaging and Ordering Details
VIBRIO CHOLERAE ANTISERA are provided as 2ml
volumes in vials with dropper attachments and
contain 0.1% sodium azide as preservative. Supplied
ready to use. This is sufficient for 50 slide
agglutination tests or 20 tube agglutination tests.
The antisera are prepared from rabbits
hyperimmunised with standard strains of V.cholerae
O1 Inaba type and Ogawa type or O139 Bengal type
organisms. All sera are heat inactivated at 56ºC for
30 minutes, absorbed to remove cross-reacting
agglutinins and filter sterilised.
Materials Required but not Provided
1. Clean glass microscope slides or glass test
tubes.
2. Chimograph or glass-pencil.
3. Disposable or platinum wire inoculation loop.
4. Suitable discard container containing 0.1%
sodium hypochlorite solution (available chlorine
approx. 1000 ppm).
5. Sterile 0.85% saline solution.
6. Autoclave capable of attaining 121ºC or a device
for heating bacterial suspensions to 100ºC.
Stability and Storage
2.
VIBRIO CHOLERAE ANTISERA should be stored at
2-8ºC and may be used until the expiry date given on
the label.
Note: allow the antiserum to freefall from the
dropper provided with the bottle. Do not
contaminate the antiserum with organism.
Do not freeze reagents.
Shelf life - 2 years from date of manufacture.
3.
Mix the reagents by tilting the slide back and
forth for 60 seconds while viewing under indirect
light against a dark background.
4.
Distinct clumping or agglutination within this
period, without clumping in the saline control
(auto-agglutination) should be regarded as a
positive result.
5.
Specimens that show agglutination only with
Inaba-type serum should be reported as
V.cholerae O1 serovar Inaba and specimens
that show agglutination only with Ogawa-type
serum should be reported as V.cholerae O1
serovar Ogawa. Specimens that show
agglutination with both types of serum should be
reported as V.cholerae O1 serovar Hikojima.
Specimens that show agglutination only with
O139 Bengal serum should be reported as
V.cholerae O139 Bengal.
Warnings and Precautions
1.
These reagents are provided for in vitro
diagnostic use only.
2.
Read instructions carefully before conducting the
test.
3.
Do not use beyond the expiry date.
4.
Wear appropriate protective clothing when
handling infectious organisms.
5.
Dispose of contaminated plasticware and
glassware by soaking in 0.1% sodium
hypochlorite solution (available chlorine
approximately 1000ppm) overnight, by
autoclaving at 121ºC for 20 minutes or more, or
according to local microbiological regulations.
Spillages should be mopped up with absorbent
material and the area swabbed with 5.0%
sodium hypochlorite solution.
6.
7.
8.
Avoid microbial contamination of opened
reagent bottles. Do not use reagents if they are
contaminated or cloudy.
Sodium azide is used as a preservative. It may
be toxic if ingested. Sodium azide may react
with lead and copper plumbing to form highly
explosive salts. Always dispose of by flushing to
drain with plenty of water.
Do not freeze the antisera. Freezing and
thawing may produce precipitation and result in
loss of activity of the reagent.
Procedures and Interpretation of Results
Cultures of organisms identified as V.cholerae by
their morphological and biochemical features may be
serotyped by the following procedures.
1.
Place two drops of sterile 0.85% saline solution
(saline) onto a carefully cleaned microscope
slide. The slide may be partitioned into several
parts using a chimograph or glass pencil. With
an inoculation loop or wire emulsify into each
drop of saline a live cell colony from a fresh agar
plate or slope culture to produce a distinct and
uniform turbidity.
Place a drop of polyvalent antiserum onto one of
the drops of emulsified isolate and to the other a
drop of saline as a control.
Note:- it should be remembered that El Tor
Vibrios cannot be distinguished from V.cholerae
O1 by serological means.
References
1.
2.
4.
5.
Sakazaki R, Tamura K, Gomez CZ, Sen R.
Serological studies on the cholera group of
vibrios. Jap J Med Sci Biol. 1970; 23: 13.
Sakazaki R, Tamura K. Somatic antigen
variation in Vibrio cholerae. Jap J Med Sci Biol.
1970; 24: 93.3.
Donovan TJ. Serology and
serotyping of Vibrio cholerae. In Vibrios in the
environment. 1984. ed Colwell RR, p83. Pub
John Wiley & Sons, New York.
Shimada T, Outbreak of Vibrio cholerae non-O1
in India and Bangladesh. Lancet 1993; 341:
1346
Cholera Working Group International Centre for
Diarrhoeal Diseases Research, Bangladesh.
Large outbreak of cholera-like disease in
Bangladesh caused by Vibrio cholerae O139
synonym Bengal. Lancet 1983; 342: 387