VIBRIO CHOLERAE ANTISERA
Liquid stable antisera for the determination of O antigens of Vibrio cholerae by slide or tube agglutination.
Introduction
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.
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 (‘rice-water’ 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.
Principle of the Test
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.
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
VIBRIO CHOLERAE ANTISERA should be stored at 2-8ºC and may be used until the expiry date given on the label.
Do not freeze reagents.
Shelf life - 2 years from date of manufacture.
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. Avoid microbial contamination of opened reagent bottles. Do not use reagents if they are contaminated or cloudy.
7. 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.
8. 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.
2. 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: allow the antiserum to freefall from the dropper provided with the bottle. Do not contaminate the antiserum with organism.
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.
Note:- it should be remembered that El Tor Vibrios cannot be distinguished from V.cholerae O1 by serological means.
References
1. Sakazaki R, Tamura K, Gomez CZ, Sen R. Serological studies on the cholera group of vibrios. Jap J Med Sci Biol. 1970; 23: 13.
2. 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.
4. Shimada T, Outbreak of Vibrio cholerae non-O1 in India and Bangladesh. Lancet 1993; 341: 1346
5. 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