Antibodies against recombinant heat shock protein 65 of Tropheryma whipplei in patients with and without Whipple's disease.

Tropheryma whipplei is the causative agent of Whipple's disease (WD), a chronic, life-threatening infection. Laboratory diagnosis is mainly based on PCR and histopathological analysis in duodenal biopsies and other specimens requiring invasive procedures. We have examined the presence of antibodies to recombinant heat shock protein (Hsp65) of T. whipplei in patients with Whipple's disease as well as in control subjects by Western blot and enzyme-linked immunosorbent assay (ELISA). A recombinant plasmid carrying the entire T. whipplei hsp65 gene was constructed, and the expression yielded a 65-kDa histidine-tagged protein. Among four patients with Whipple's disease, two showed an IgG- and one an IgA-response, respectively, when analyzed by Western blotting, whereas from 10 patients without Whipple's disease, only two patients showed a positive IgG-response. The differences between the sera from patients and controls were thus not significant. Successful purification of the protein was achieved by Ni-NTA affinity chromatography. Quantitative analysis of serum antibodies by ELISA demonstrated that antibody levels in the sera of 14 patients were not significantly higher than in those of 89 control subjects. The established ELISA test is not useful to clinical diagnostics.

Tropheryma whippelii DNA in saliva of patients without Whipple's disease.

Tropheryma whippelii is the causative agent of Whipple's disease, a difficult to diagnose systemic illness. Amplification of part of its 16S ribosomal RNA gene(s) has become a standard diagnostic method because of increased sensitivity as compared to classical histopathological analysis. Recently, we demonstrated the presence of T. whippelii DNA by PCR in duodenal biopsies and/or gastric juice of a considerable fraction of individuals without clinical signs of Whipple's disease. In this follow-up study, saliva and dental plaques of the same patients were screened for the presence of T. whippelii DNA. Six out of the 14 previously PCR-positive persons but none of the 17 controls had T. whippelii DNA in their saliva. Our results suggest that Whipple bacteria are ubiquitous environmental or commensal organisms causing Whipple's disease only in a particular subset of individuals, possibly those with an as yet uncharacterized immunological defect.

Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of "Tropheryma whippelii" and its use for detection of "T. whippelii" in clinical specimens by PCR.

Using broad-spectrum primers, we have amplified, cloned, and sequenced a 620-bp fragment of the "Tropheryma whippelii" heat shock protein 65 gene (hsp65) from the heart valve of a patient with Whipple's endocarditis. The deduced amino acid sequence shows high similarity to those from actinobacteria, confirming that "T. whippelii" is indeed a member of this phylum. Based on the nucleotide sequence, we have developed a "T. whippelii"-specific seminested PCR. Seventeen patients shown to be positive by 16S ribosomal DNA (rDNA) PCR and/or internal transcribed spacer (ITS) PCR were also positive by hsp65 PCR. All 33 control specimens from patients without Whipple's disease and negative for "T. whippelii" by both seminested 16S rDNA and ITS PCR remained negative. All amplicons digested with XhoI revealed an identical restriction pattern. Eight of the 17 hsp65 amplicons representing all three previously described ITS types were sequenced. Three of the amplicons showed slight differences, but none of the mutations detected affected the amino acid sequence of the corresponding protein. We conclude that the hsp65 gene is a suitable target for the specific detection of "T. whippelii." Its product represents a putative antigen for a future serodiagnostic assay.