Salmonella Serotyping

The sample control microbiological laboratory has accredited the method HRI CEN ISO/TR 6579-3:2014 guidelines for serotyping of Salmonella spp. The reference method of serotyping with antisera is a continuation of the horizontal method of proving the presence of Salmonella spp. therefore, it largely depends on good preparation, homogenization of samples and enrichment and isolation procedures on nutrient media. In biochemically confirmed samples, the serotyping procedure is carried out independently of the sample from which it was isolated. Pursuant to Commission Regulation (EC) no. 2073/2005 on microbiological criteria for food stipulates that fresh poultry meat obtained from breeding flocks of the Gallus gallus species, laying hens, fattening chickens and flocks of breeding and fattening turkeys and carcasses of poultry, broiler chickens and turkeys in which Salmonella spp. have been detected must serotype to determine the absence of Salmonella serovar Enteritidis and Salmonella serovar Typhimurium. Therefore, it is important that the procedure is carried out only in adequately equipped laboratories, under expert supervision with respect to all precautionary measures, following the manufacturer's instructions when handling antisera and adequate disposal of incubated materials.

According to the scheme developed by Kaufmann and White, the presence or absence of specific O-antigens, H-antigens and Vi-antigens in the isolate is determined. More than 2,600 different serovars are known today.

Antigenic formula of the five most important Salmonella serovars associated with public health

Antigens that appear only if the culture is lysogenized with the converting phage are always underlined, while some antigens may be present or absent, independent of the phage, and are written in square brackets.

O-antigen (somatic antigen)

It consists of components of the cell wall of bacteria, and contains lipopolysaccharide molecules, which are endotoxins of gram-negative bacteria. It consists of lipid A and polysaccharides. All toxicity is associated with lipid A, and the polysaccharide is somatic or O-antigen. The reaction of O-antigen with antisera results in granular agglutination. Based on the somatic antigen they possess, they are classified into serological groups, which are indicated by letters starting with group A, which includes the O:2 antigen, up to group Z, which contains the O:50 antigen. As there are more O-antigens than letters of the alphabet, the remaining antigenic groups are designated by their O-antigen numbers, from O:51 to O:67. Nowadays, it is preferable to mark with the number of O-antigens characteristic of the group. Letters are retained and are shown in brackets, eg O:4 (B).

H-antigen (flagellar antigen)

Flagella or flagella of bacteria contain flagellar antigen, the main component of which are proteins. It can be easily decomposed by alcohol, acid and temperature above 60 °C. The reaction of H-antigen with antisera results in fluffy agglutination (softer than granular). Many possess two phases of the H-antigen, but monophasic and triphasic variants are also known. The first phase is called the specific phase and the second is the non-specific phase. The first phase is marked with a lowercase letter from a to z. Since many new H-antigens have been discovered, they have a numerical designation in addition to the letter z, for example z1, z2, z3 ... z91.

Vi-antigen (capsular antigen)

Capsular (surface) antigen can cover O-antigens so that bacteria do not agglutinate with O-antisera. The main component of the Vi-antigen is a polysaccharide. Strains possessing Vi-antigen are more virulent than strains without Vi-antigen. Vi-antigen can be present in only three serovars Salmonella Typhi, Salmonella Paratyphi, Salmonella Dublin.

Serotyping procedure

The bacterial culture must be pure, biochemically confirmed and inoculated onto a non-selective nutrient medium. Each antiserum supplier produces its own antiserum kits with its own unique instructions for use, so it is important to follow the supplier's instructions for optimal results. Before starting agglutination, it should be checked on the slide with antisera that the isolate to be serotyped spontaneously agglutinates. One drop of the appropriate antiserum is placed on a clean slide, the culture from the agar is applied directly to the drop of antiserum on the slide and mixed gently until the suspension becomes homogeneous. With light movements of the slide, the suspension is stirred for no more than a minute, and the agglutination is examined with the naked eye or a magnifying glass (on a dark background).

Agglutination with O-antigens

The procedure usually starts with appropriate antisera containing specific antibodies for multiple somatic antigens. There are antisera that contain antibodies for 4 to 7 somatic antigens, for example polyvalent antiserum for O-antigens A, B, D, E and L, then for C, F, G, H O-antigens, etc. Polyvalent antisera are used to use fewer procedures agglutination determines which serological group the isolate to be typed belongs to. When it is determined with which polyvalent antiserum the isolate agglutinates, the procedure continues with agglutination with monovalent antisera. For example, if the isolate agglutinated with polyvalent antiserum for serological groups C, F, G and H, the procedure continues with monovalent antisera for the four mentioned groups.

Agglutination with H-antigens

When it is determined to which serological group the isolated strain belongs, agglutination with H-antisera is used to determine the exact serovar of Salmonella spp. A large number of Salmonella serovars are biphasic, which means that its flagella have two phases: phase 1 (specific) and phase 2 (non-specific). A cultivated culture of Salmonella can have both or only one of the phases. If a serovar with two phases produces a positive reaction with one phase but not with the other, the phase inversion procedure must be used. Antiserum of the phase that has been successfully agglutinated is added to the semi-soft Sven gard agar, which allows Salmonella to crawl on the surface. This blocks the development of that phase, and the tested isolate can crawl only if it develops the second flagella phase. 1-2 drops of antiserum for the flagella phase that has been successfully agglutinated are poured into a small Petri dish, for example, if an isolate with H:i antiserum has been successfully agglutinated in Salmonella Typhimurium, then this antiserum is added to the Petri dish. Sven Gard's agar (cooled to 47°C) is added to the diluted antiserum, the agar and antiserum are gently mixed in a Petri dish and left to harden. After that, the desired culture is inoculated in the middle of the Petri dish and incubated for 24±3 hours. It is important that the next day the isolate from the center where it was inoculated crawls over the surface of the agar in the Petri dish. Then, the part that has crawled is taken by ezo and agglutinated with a certain antiserum (in the case of S.Typhimurium with H:1,2 antiserum which is the second phase).

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