1124

Ouchterlony immunodiffusion analysis of the relationship ofCjejunitoxin (CJT) to E coli heat-labile toxin (LT) and cholera toxin (CT)with antisera to LT (cr LT) and CT (a CT).

ponent of GM1 ganglioside. C jejuni strain INN-73-83 (kindlyprovided by Dr L. Ruiz-Palacios) was grown at 42°C for 24 h in GCmedium (Difco) supplemented with 0. 1°70 ’IsoVitaleX’ (BBLMicrobiology Systems) under agitated conditions in the presence of8% CO2 and treated for the final 10 min with 2 mg polymyxin perml. The sterile broth filtrate was concentrated 20-fold by sequentialpassage through 100 000 and 10 000 molecule weight cutoff ultra-filtration membranes and precipitated with 70% ammonium sul-phate. After dialysis against TEAN buffer ("tris"/EDTA/sodiumazide/sodium chloride), the precipitate was passed through a BiogelA I - 5 m agarose column (Biorad) and eluted with galactose asdescribed for the purification of LT. The protein present in thegalactose front was dialysed against TEAN buffer and concentratedby ultrafiltration.These purification procedures resulted in a nearly 23 000-fold

increase in the specific activity of CJT; values in the CHO assay,expressed as ng CJT activity per mg protein, were 27 for the brothsupernatant and 625 000 for the purified toxin preparation. Thesmallest amount of pure toxin that gave a positive response in theCHO assay was CJT 40 pg, LT 40 pg, and CT 80 pg; the activity ofeach toxin was abolished by exposure to 70°C for 2 h or 96°C for 10min. Ouchterlony immunodiffusion analysis showed lines of

identity between CJT and LT with antiserum to LT and partialidentity between CJT and CT with antiserum to CT (figure). In theCHO assay, 1 ml of antiserum to LT neutralised 320 ng of LT, 160ng CJT, and 160 ng CT, while 1 ml of antiserum to CT neutralised2048 ng CT, 160 ng CJT, and 160 ng LT. Based on comparison oftoxin concentrations that yielded an optical density of 0 - 600 at 405nm in a single species double sandwich ELISA using affinitycolumn purified antiserum to LT, CJT had 65% and CT 48%homology with LT.Although it remains to be established, it seems likely that entero-

toxin production will prove to be a contributory factor in the patho-genesis of the secretory type of diarrhoea caused by Cjeuni that is socommon among children living in developing countries.6 Ourobservations with pure CJT indicate that it is immunologicallyrelated to the cholera/coli group of heat-labile, adenylate cyclaseactivating enterotoxins. Various toxoid vaccines against members ofthis group are under development; the partial immunologicalhomology of CJT with this group raises the possibility that one ofthese toxoid vaccines may also affect C jejuni toxin mediatedsecretory diarrhoea.

Department of Medicine,School of Medicine,University of Rochester,Rochester, NY 14642, USA

F. A. KLIPSTEINR. F. ENGERT

5. Clements JD, Finkelstein RA. Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures InfectImmun 1979, 24: 760-69.

6. Bokkenheuser VD, Richardson NJ, Bryner JH, Roux DJ, Schutte AB, Koornhof HJ,Freiman I, Hartman E Detection of enteric campylobacteriosis in children. J ClinMicrobiol 1979, 9: 227-32

7. Levine MM, Kaper JB, Black RE, Clements ML New knowledge on pathogenesis ofbacterial enteric infections as applied to vaccine development. Microbiol Rev 1983;47: 510-50.

FATAL POST-SPLENECTOMY SEPSIS DESPITEPROPHYLAXIS WITH PENICILLIN AND

PNEUMOCOCCAL VACCINE

SIR,-Measures to reduce the risk of fatal infection after

splenectomy include long-term antibiotic prophylaxis with

penicillin and the anti-pneumococcal vaccines. ],2 These measuresmay not always be effective.At the age of 3 months a baby was admitted to -hospital for

haemolytic anaemia with massive splenomegaly, due to a congenitalcytomegalovirus infection. Corticosteroid treatment was

unsuccessful and he showed a poor response to transfusion.

Splenectomy was done in January, 1981, when he was 2 years old.Penicillin 125 mg twice daily was prescribed, and later increased to250 mg twice daily. He was immunised with antipneumococcalvaccine (’Pneumovax’) in December, 1981. 2 years after operation,he was admitted to hospital unconscious after a vague illness of 2days’ duration, with pyrexia, vomiting, and malaise. He had otitismedia and meningitis with pneumococcus type 19. He did not

respond to treatment and died 5 days later. No necropsy was done.An underlying immune defect was suspected but not proved. His

mother had recovered from an illness of over 10 years’ durationcharacterised by lymphadenopathy and splenomegaly. Repeatedbiopsy samples showed plasma cells and histiocytes: Hodgkin’sdisease was suggested but the general opinion was that this was anon-specific reaction. The boy was found to be excretingcytomegalovirus in the urine at the age of 3 months, and thispersisted up to 16 months. There was only a low titre of

anticytomegalovirus antibodies of 1:20 on repeated tests, and nopoliovirus antibodies despite immunisation. Serum

immunoglobulin levels were repeatedly normal (eg, IgG 55, IgA 26,IgM 90, IgE <5iU/ml), and lymphocyte markers were normal (anti-T 59%, sIg 10%, phytohaemagglutin response 18 900 counts/minwith a total blood lymphocyte count of 2-4x10/1). A furtherunusual feature was the development of a prominent generalisedlymphadenopathy after splenectomy, together with a

lymphocytosis of up to 35’ 96 x 109/1 with morphologically normalsmall lymphocytes, shown to be 35% B cells, 19% positive with anti-kappa and 16% positive with anti-lambda. 35% reacted with anti-IgM, 3007o with anti-IgD, 307o with anti-IgG, and 907o with anti-IgA.This polyclonal reaction was attributed to infection rather than toneoplasia; it seems probable that the lymph nodes had enlarged tocompensate for the absent spleen.In this case the pneumococcus was type 19, which is included in

the vaccine used. However, vaccination was done after the

operation, when the patient was nearly 3 years old. Vaccination aftersplenectomy produces a’poorer response than it does when givenwith the spleen present,3,4 and it is less effective in young children,particularly under the age of years.5,6 Another reason for failure ofprophylaxis in this case is the poor antibody response demonstratedby the child, although specific pneumococcal antibodies were notmeasured.

Infections and deaths from pneumococcal sepsis in vaccinatedchildren or after prophylactic penicillin have been reported, but thisis the first case known to me where fatal pneumococcal meningitishas followed dual prophylaxis with both antipneumococcal vaccineand penicillin.

I thank Dr G. V. Feldman for referring the case and Dr T. N. Stanbndge forfurther information.

Royal Manchester Children’s Hospital,Manchester M27 1HA D. I. K. EVANS

1. Ammann AJ Current status of pneumococcal polysaccharide immunisation in patientswith sickle cell disease or impaired splenic function Am J Pediatr Hematol Oncol1982, 4: 301-36.

2. Ferguson A Hazards of hyposplenism. Br Med J 1982; 285: 1275-76.3. Hosea SW, Brown EJ, Burch CG, Berg RA, Frank MM. Impaired immune response of

splenectomised patients to polyvalent pneumococcal vaccine. Lancet 1981, i:

804-07.4 Di Padori F, Dung M, Wadstr&ouml;m J, Harder F Role of spleen in immune response to

polyvalent pneumococcal vaccine Br Med J 1983; 287: 1829-32.5. Karup Pedersen F, Henrickson J, Schiffman G Antibody response to vaccination with

pneumococcal capsular polysaccharides in splenectomised children. Acta PaediatrScand 1982; 71: 451-55

6 Lawrence EM, Edwards KM, Schiffman G, Thompson JM, Vaughan WK, WrightPF. Pneumococcal vaccine in normal children. Am J Dis Child 1983, 137: 846-50