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Yersinia enterocolitica IgG/IgA Westernblot

 

Immunoblot for the detection of IgG- or IgA-antibodies against Yersinia enterocolitica in serum

 SUMMARY AND EXPLANATION OF THE TEST

Yersiniae are members of the genus Enterobacteriaceae.

There are three different species:

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Y. pestis

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Y. pseudotuberculosis

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Y. enterocolitica

On the basis of the O-antigens, 34 serotypes can be differentiated. Only serotypes containing a plasmid can infect humans (in Europe: 0:3, 0:9, 0:5, 27). About the transmittance of the disease there are assumptions but no proof.

In most cases, a Yersinia enterocolitica infection causes mesenterial lymphadenitis. The clinical picture can be enteritis, pseudoappendicitis, ileitis or colitis. The severity of the illness depends on the age of the patient. Extramesenterial forms (20 – 25 % of all cases), local infections after dissemination, septic forms and the lymphadenopathic syndrome can occur with or without a preceding enteritis. The persistence of Yersinia enterocolitica in lymph nodes and the intestine associated lymphatic tissue is probably responsible for chronic and recurrent forms.

The most frequent secondary immunopathologic clinical pictures caused by Yersinia are reactive arthritis and Erythema nodosum.

The Yersinia-induced Reactive Arthritis normally begins 1 – 4 weeks after the onset of the intestinal symptoms. But it is remarkable that a large number of patients cannot report anamnestic intestinal symptoms before the arthritic symptoms. In most cases, the joints of the legs and feet are affected. After 3 - 12 months the disease is normally healed, but in some patients it proceeds into a chronic or recurrent stage, which cannot be distinguished from other forms of spondylarthritis. 60 – 80 % of the arthritis patients are HLA-B27-positive. In most cases of Yersinia-induced arthritis, persistent Yersinia-specific IgA antibodies or  immunocomplexes can be found in sera or synovia, further a Yersina-specific proliferation of synovial lymphocytes. The IgA-persistence is associated with the persistence of pathogenic (plasmid-containing) Yersiniae in intestinal lymphatic tissue.

In some cases of Yersinia infections an Erythema Nodosum can be found. It is more frequent in adult women and can be accompanied by reactive arthritis. The skin symptoms can occur 4 - 6 weeks after infection.

The old serological methods for the detection of Yersinia antibodies are based on agglutinins. But in most cases these agglutinins are below a significant detection level at the onset of the arthritic symptoms. In addition, examinations of stool samples are negative at this time. So most of the Yersinia-infections cannot be diagnosed. A further problem of the old methods like Widal-reaction is the cross reactivity to other gram-staining negative bacteria. Only the immunoblot technique using specific virulence associated antigens, and the accompanied differentiation and detection of class-specific antibodies, can help to answer the question whether there is a recent, a persistent or an earlier infection with Yersinia enterocolitica. The answer is very important for a prognosis of the disease and a decision on therapy.

REFERENCES

  1. Heesemann, J., Gross, U., Schmidt, N., Lauffs, R. Immunochemical analysis of plasmid encoded

  2. proteins released by enteropatogenic Yersinia sp. grown in calcium deficient medium. Infect. Immunol. 54, 561-567 (1986)

  3. Stahlberg, T. H., Granfors, K., Toivanen, A. Immunoblot analysis of human IgG, IgM and IgA response to plasmid encoded antigens of Yersinia enterocolitica serovar 0:3. L. Med. Microbiol, 24, 157-163 (1987)

  4. Heesemann, J., Eggers, C., Schröder, J. Serological diagnosis of yersiniosis by immunoblot

  5. technique using virulence-associated antigen of enteropathogenic Yersiniae. Contrib. Microbiol. Immunol. 17, 285-289 (1987)

  6. Heesemann, J., Kalthoff, H., Koch, F. Monocional antibodies directed against plasmidencoded

  7. released proteins of enteropathogenic Yersiniae. FEMS Microbiol. Lett. 36, 15-19 (1986) Heesemann, J., Gaede, K. Mechanisms involved in the pathogenesis of Yersinia infections.

  8. Rheumatol. Int. 9, 213-217 (1989)

 

ANALYSIS OF BAND PATTERN AND INTERPRETATION OF THE RESULTS

For this immunoblot the supernatants of calcium-deficient Yersinia-cultures serovar 0:9 were used. Under these conditions, the human pathogenic Yersinia shed, so called „Yersinia outer proteins“ (yop) or „release proteins“ (RP).

These proteins are encoded on a plasmid, they are highly antigenic and very specific for human pathogenic Yersinia. Against the following proteins an immunoresponse can be observed:

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Yop 1 (58 kD)

About this protein nearly nothing is known. Possibly it is involved in the active iron intake from the environment. In most cases IgG-antibodies can be detected.

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Yop 2a (48 kD)

This protein is responsible for the ability of Yersiniae to inhibit the aggregation of thrombocytes. The immunoresponse is mostly IgG.

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Yop 2b (46 kD)

This protein makes the Yersiniae resistent to phagocytosis. Immunoresponse mostly IgG.

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Yop 3 (40 kD) or V-Antigen

Little is known about the role of this protein. Immunoresponse: IgG or IgA.

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Yop 4a (36 kD)

The function of this protein is disputed. The major IgA response is directed against this protein. Therefore the interpretation of the results is especially important.

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Yop 4b (34kD)

Function not clear, immunoresponse IgA or IgG, but labelled only in rare cases.

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Yop 5 (25 kD)

This protein plays an important role in the cytotoxicity of the Yersiniae. The immunoresponse is IgG in most cases.

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„sp - Protein“ (80 kD)

This is no plasmid encoded protein, but seems to be specific to Yersinia.

Interpretation

Cut off - Standard setting: IgA - 10 %, IgG - 15 %

The results of this immunoblot are scored Yersinia-IgG positive when at least 4 of the YOP bands are detected, IgG borderline when 3 of the YOP bands are detected.

The results of this immunoblot are scored Yersinia-IgA positive when at least 2 of the YOP bands are detected.

Further assistance in interpretation:

Persistant Yersinia-specific IgA antibodies are usually detected in patients suffering from Yersinia-induced arthritis. Additional antibody reactions in the immunoblot, such as heat shock proteins (at the moment are not characterized), point to a chronic inflammation (especially an IgA response).

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