Immunoelectrophoretic characterization and cross-reactivity of Rochalimaea henselae, Rochalimaea quintana and Afipia felis.

The soluble antigens of Rochalimaea henselae, Rochalimaea quintana and Afipia felis were characterized by crossed immunoelectrophoresis using bacterial sonicates as antigens against pooled hyperimmune rabbit sera. A precipitin pattern was drawn for each bacterium and shown to be reproducible and stable even when normal or preimmune rabbit serum was incorporated in the intermediate gel. By this technique 56 antigens were identified from R. henselae, 49 from R. quintana, and 39 from A. felis. The serological cross-reaction between R. henselae, R. quintana and A. felis, and between these 3 bacteria and 32 pathogenic bacteria was analysed by rocket-line immunoelectrophoresis, crossed-line immunoelectrophoresis, and tandem-crossed electrophoresis. It was concluded that (i) 4-7 antigens distinguish R. henselae, R. quintana and A. felis from each other, (ii) both Gram-positive and Gram-negative bacteria cross-react with R. henselae, R. quintana and A. felis antisera, (iii) the cross-reacting antigens of Gram-negative bacteria have both precipitating and non-precipitating specificities, whereas Gram-positive bacteria have mainly non-precipitating specificities, (iv) the cross-reacting antigens are common to several species, and (v) fewer cross-reacting antigens are found in phylogenetically disparate species than in more closely related species.

Antibody response in rabbits infected with Rochalimaea henselae, Rochalimaea quintana and Afipia felis.

Antibody responses in three pairs of rabbits inoculated with live Rochalimaea henselae, Rochalimaea quintana and Afipia felis were studied by enzyme immunoassay with whole-cell and bacterial sonicates as antigen. No differences in measured antibody responses were found with the two types of antigen preparation. Two rabbits did not respond with antibody production. In the remaining rabbits there was a low-titred antibody response that showed no significant cross-reaction with related bacteria. After rechallenge the antibody response rose significantly and there was significant cross-reaction with related bacteria. The antigens involved in the antibody response were examined by crossed immunoelectrophoresis. After the initial inoculation 5-7 precipitin lines of the reference diagrams were deflected, including lines which cross-reacted with antigens found in related bacterial species. After reinoculation several more precipitin lines were deflected, including additional lines cross-reacting with antigens present in related bacteria and common bacterial antigens.

Production and characterization of mouse monoclonal antibodies against Afipia felis.

A series of 10 monoclonal antibodies reacting with Afipia felis antigens were selected from mice immunized with live organisms of the reference strain ATCC 53690. Immunoblotting against SDS-PAGE-separated A felis sonicate allowed the antibodies to be classified into three groups: 1) 168-4, -6, -7 and -10 reacted with a 53 kDa antigen, 2) 168-1, -3 and -9 reacted with both 53 kDa and 60 kDa antigens, and 3) 168-2, -5 and -9 reacted with other antigens. Antibodies of group 1 did not cross-react with other Afipia species or 36 unrelated bacteria, whereas those of groups 2 and 3 reacted with other Afipia species and some unrelated bacteria. Immunoblots of crossed immunoelectrophoretic patterns of A. felis sonicate against rabbit antiserum showed that antibodies of groups 1 and 2 bound to the same precipitin arcs. Antibodies of group 1 reacted with a species-specific epitope on the 53 kDa antigen, while those of group 2 reacted with other epitopes shared by the 53 kDa and 60 kDa antigens. The binding of antibodies of group 1 to A. felis sonicate was inhibited by post-infection rabbit serum, whereas no inhibition was observed for antibodies of group 2. The species-specific epitope of the 53 kDa antigen and the early appearance of antibodies against this epitope after infection suggest that this antigen can be used in a serodiagnostic test for A. felis infection.

Identification of Afipia felis antigens in culture medium: reaction with human sera.

Fourteen protein antigens were identified on SDS-PAGE of Afipia felis culture supernatant. Immunoblotting against 10 monoclonal antibodies obtained from mice infected with live A. felis showed that 4 antibodies reacted with a 56 kDa band and 3 with both 56 kDa and 62 kDa bands. Compared with A. felis sonicate, the reacting proteins in culture supernatant showed an increase in molecular mass of 2-3 kDa, suggesting that they were more glycosylated. Purified antigen obtained by affinity chromatography of culture supernatant on the seven immobilized antibodies was tested against antibodies reacting with the 56 kDa and 62 kDa bands. All eluates contained both components, suggesting that the antibodies were directed against different epitopes of a double antigen held together during the affinity chromatography but cleaved by reduction and SDS-PAGE. The molecular size of the uncleaved protein in culture supernatant was determined by size-exclusion chromatography as > 1000 kDa. Testing of pre- and post-infection rabbit sera in immunoblotting against culture supernatant demonstrated that the 56 kDa and 62 kDa components gave the most prominent specific reactions with post-infection sera. One of fifty human sera submitted for testing for cat-scratch disease and 1 of 50 sera from healthy blood donors reacted with several bands in A. felis culture supernatant, including the 56 kDa and 62 kDa bands.

Immunopurified extracellular Bartonella henselae antigen for detecting specific antibodies by enzyme immunoassay.

Protein antigens of Bartonella henselae bacterial sonicate supernatant and concentrated cell-free culture filtrate were examined by SDS-PAGE. The sonicate supernatant gave 38 bands and the culture filtrate at least 21, of which 18 were of bacterial origin. Immunoblotting against 13 monoclonal antibodies obtained from mice infected with live B. henselae showed that 10 of these antibodies reacted with a narrow 225 kDa band and varying smears of bands ranging from 36 to 240 kDa in the sonicate, but only with a single 200 kDa band in the culture filtrate. Testing of pre- and post-infection rabbit sera in immunoblotting against culture filtrate demonstrated that the 200 kDa component gave the most prominent specific reaction with post-infection sera. The 200 kDa antigen was isolated by immunoaffinity chromatography of concentrated culture filtrate, and its molecular size determined by size-exclusion chromatography as > 1000 kDa. The immunopurified antigen was compared with bacterial sonicate as coating antigen in EIA for determining humoral immune responses in rabbits inoculated with live B. henselae. The two antigens gave almost identical results for IgM and IgG responses. The specificity of the immunopurified antigen was tested in EIA against hyperimmune rabbit sera and sera of rabbits inoculated with live B. henselae, B. quintana and Afipia felis. Only the hyperimmune serum against B. henselae and the sera of the rabbits inoculated with live B. henselae reacted with the immunopurified antigen, whereas the B. henselae sonicate cross-reacted with hyperimmune and post-infection sera of rabbits inoculated with B. quintana and A. felis.

A new technique for Gram staining paraffin-embedded tissue.

Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining.

Acridine orange fluorescence techniques as alternatives to traditional Giemsa staining for the diagnosis of malaria in developing countries.

Traditional Giemsa-stained thick blood films were compared with 2 fluorescence microscopy techniques, acridine orange (AO) staining of thin blood films and the quantitative buffy coat (QBC) method, for the microscopical diagnosis of malaria. Of 200 samples examined, 141 were positive by Giemsa staining, 146 by AO and 137 by QBC. Overall sensitivities for the 2 fluorescence techniques compared to Giemsa staining were good: AO 97.9% and QBC 93.6%. However, with parasitaemias < 100/microL the QBC sensitivity fell to 41.7% whereas that of AO was 83.3%. Both AO and QBC were unable to differentiate accurately between individual malaria species. We conclude that the QBC technique alone cannot replace Giemsa-stained thick blood films for most purposes in an African setting. However, apart from species differentiation, the AO method is an appropriate technique for the laboratory diagnosis of malaria in developing countries.

[Benefit, effectiveness and cost of parasite investigations in the county of Copenhagen]. / Nytte, effektivitet og udgift til parasitundersøgelser i Københavns Amt.

OBJECTIVE: To evaluate the cost and effectiveness of parasite investigations in our laboratory in order to restrict the number of specimens. METHODS: The analysis were performed retrospectively on 53,701 specimens submitted for cysts, ova, and larvae, 224 specimens for intestinal trophozoites, 1,888 cellophane tape impressions, 3,134 malaria smears, and 524 bronchial aspirates. RESULTS: Potentially pathogenic parasites were demonstrated in 3.2% of specimens examined for cysts, ova, and larvae, 6% examined for intestinal trophozoites, 16% for pinworm, 7% for malaria, and 9% for Pneumocystis. The cost of demonstrating a single infection with a pathogenic parasite was calculated to be DKK 6206 for cyst, ova, and larva specimens. This cost could be reduced considerably, if the examination of patients with acute diarrhoea, but without a history of travel was omitted. Similarly, the demonstration of trophozoites, pinworm, malaria and Pneumocystis in a person was calculated to be DKK 1122, DKK 526, DKK 2874 and DKK 3716, respectively. Examination of three specimens for cysts, ova, and larvae showed 62-74% of the infections in the first sample. Two specimens increased the frequency of positive findings by 13-23% and three specimens by a further 9-12%. For the blood smears, 92% of the infections were demonstrated in the first sample. DISCUSSION: The analysis shows that to obtain an acceptable ratio between cost and diagnostic yield, the indication for the investigations should be tightened up. However, examination of three samples from each patient is necessary to exclude a parasitic infection.

[Cat-scratch disease and bacillary angiomatosis. An old and a new infectious disease with common etiology?]. / Kattekradssygdom og bacillaer angiomatosis. En gammel kending og en ny infektionssygdom med faelles aetiologi?

A review of cat-scratch disease (CSD) and bacillary angiomatosis (BA) is presented on the basis of published articles. Two newly identified bacteria--Rochalimaea henselae and Afipia felis--have been isolated from patients with CSD. Preliminary investigations seem to indicate that A. felis is an uncommon cause of the disease. CSD may appear as a local suppurative lymphadenopathy or a systemic infection. BA is caused by Rochalimaea species and may appear as cutaneous, mucous or visceral angiomas or bacteremia. It may be a special manifestation of CSD in immunocompromised patients. A description is given of the various pathological pictures and differential diagnosis, and an evaluation is made of the different diagnostic methods, namely visualisation of bacteria in the lesions with Warthin-Starry's silver impregnation, isolation of bacteria, demonstration of bacteria with gene technique and detection of antibodies. The treatment of the disease is discussed.

[Perinaud's oculoglandular syndrome as a manifestation of cat-scratch disease]. / Parinauds okuloglandulaere syndrom som en manifestation af kattekradssygdommen.

The first Danish case of Parinaud's oculoglandular syndrome, a manifestation of cat-scratch disease, is reported in a 35-year-old man presenting with an enlarged preauricular lymph node and an ipsilateral conjunctival granuloma. Surgical removal of the granulomatous lesion was followed by rapid healing. The diagnosis was verified by demonstrating a high antibody tire against Rochalimaea (Bartonella) henselae. On subsequent questioning the man gave a history of acquiring a kitten six weeks before his illness. The importance of eye examination in patients presenting with preauricular lymphadenopathy is emphasized.

A case with severe diarrhoea and Strongyloides stercoralis infection.

A case with severe diarrhoea and Strongyloides stercoralis infection is described. Further examination showed that the patient also had abnormal colonization of the duodenum with Hafnia alvei and that this disappeared when the Strongyloides infection was treated with mebendazole. Symptoms such as abdominal pain, diarrhoea, "skin rash" and malabsorption in association with blood eosinophilia should arouse suspicion of strongyloidiasis.

A survey of helminths in stray cats from Copenhagen with ecological aspects.

At autopsy of 230 adult stray cats, 120 from backyards and 110 from gardens, the intestinal tract was scrutinized for helminths. The prevalence of Toxocara cati, Taenia taeniaeformis and Dipylidium caninum was found to be 79%, 11% and 14%, respectively. Comparisons were made with the results of previous Danish investigations. The prevalence of Toxocara cati was found to be independent of time of collection and the sex and habitat of the cats and identical in cats with or without Taenia. This indicates that paratenic hosts do not play an important epizootiological role in the transmission of T. cati. The intensity of Toxocara per cat followed a negative binomial pattern. The high prevalence of T. cati combined with most cats having a low wormload shows that the cat population generally possesses a high degree of resistance against superimposed infections. The intensity of male Toxocara increases with the size of the worm population. This we consider to be an expression of increasing susceptibility of the cats. The prevalence of T. taeniaeformis was significantly higher in garden cats, due to their greater opportunity for catching mice. D. caninum, however, was significantly more frequent in backyard cats, probably owing to better living condition for the flea larvae in backyards. For both T. taeniaeformis and D. caninum a higher frequency was found in female cats, which is thought to be associated with their care for the kittens.