A Agtll cDNA library was constructed from poly(U)-Sepharose-selected Entamoeba histolyica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castelanui, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolylica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically -"S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-12MI-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4?C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

We determined MICs of 20 antimicrobial agents for 50 representative strains of four subgroups of Campylobacter-like organisms (CLOs) by agar dilution. Ampicillin, gentamicin, doxycycline, tetracycline, ceftriaxone, rifampin, spectinomycin, nalidixic acid, and chloramphenicol were active against all strains of CLOs. Most CLO strains (83%) were inhibited by 4 ,ug of sulfamethoxazole per ml and by 8 ,Ig of trimethoprim-sulfamethoxazole per ml. Of type 1 strains, 28% were resistant to 8 ,g of erythromycin per ml. In addition, cross resistance between erythromycin and clindamycin was always present. Type 1 strains exhibited a broad distribution of MICs of metronidazole and streptomycin, whereas all type 2 strains were uniformly susceptible to metronidazole and resistant to streptomycin. Unlike type 1 and 3 strains, type 2 CLOs were susceptible to cephalothin and penicillin G and highly resistant to streptomycin. The type 3 strain was uniquely resistait to cefazolin. The majority of strains were not inhibited by cefoperazone; and all were resistant to trimethoprim. In contrast to Campylobacterjejuni and Campylobacterfetus subsp. fetus, ail CLOs tested were susceptible to 0.5 ,ug of rifampin per ml.Increased awareness of the role of Campylobacter spp. in human disease and the development of improved selective media have resulted in recent isolation of several novel Campylobacter spp. from humans (7,8,10,13,14,(16)(17)(18). Among these, we isolated Campylobacter-like organisms (CLOs) from symptomatic and asymptomatic homosexual nmen being evaluated for enteritis, proctitis, and proctocolitis in a sexually transmitted diseases clinic. These organisms were isolated by using, a selective medium containing vancomycin, polymyxin B, trimethoprim, and amphotericin B supplemented with 10%o sheep blood (2) after incubation at 37°C in a microaerophilic environment. The classification of these CLOs within the genus Campylobacter and their phenotypic characterization into three subgroups were recently described by Fennell et al. (8). Further studies revealed four genetically distinct CLO subgroups (types 1A, 1B, 2, and 3), two of which (types 1A and 1B) were phenotypically identical (21a). The spectrum of diseases associated with CLO infection includes asymptomatic gastrointestinal colonization (17), proctocolitis (17)

We isolated "Campylobacter hyointestinalis" from the rectal culture of a homosexual man with proctitis. Phenotypic characterization of the isolate was confirmed by DNA hybridization by using the taxonomic spot blot. "C. hyointestinalis" was previously isolated only from animals but should be considered in the identification of Campylobacter species isolated from humans.

Monoclonal antibodies to Naegleria fowleri and Acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured N. fowleri and Acanthamoeba spp. Four monoclonal antibodies to N. fowleri were specific for N. fowleri and had no reactivity to A. polyphaga. Similarly, four monoclonal antibodies to A. polyphaga did not react with N. fowleri. Two of the four monoclonal antibodies to A. polyphaga did not react with other Acanthamoeba spp. tested, while two of the antibodies demonstrated a high degree of cross-reactivity with a putative Acanthamoeba castellanii strain by immunofluorescence microscopy; this was confirmed by fluorescence flow cytometry for one of the antibodies. These monoclonal antibodies were used to identify Acanthamoeba trophozoites in infected brain sections of a patient who died of suspected Acanthamoeba-caused granulomatous amoebic encephalitis, demonstrating potential utility in the direct identification of N. fowleri and Acanthamoeba spp. in clinical specimens.

The 29 kDa protein of pathogenic Entamoeba histolytica is a cysteine-rich surface antigen which we recently characterized by cDNA sequencing and by using monoclonal antibodies which differentiated between pathogenic and non-pathogenic clinical isolates. To determine the structure and biochemical attributes of this protein, a repertoire of immunological techniques using monoclonal antibodies, and radiolabelling were employed. We demonstrated that the 29 kDa protein forms a 60 kDa dimer and a high-molecular-mass oligomer(s) on the surface of the organism through disulphide bonds, and is the major accessible free thiol-containing surface protein of E. histolytica. The deduced amino acid sequence encoding the 29 kDa protein was found to share a common amino acid domain with sequences reported for Helicobacter pylori, Salmonella typhimurium, MER5 gene expressed in murine erythroleukemia cells, Clostridium pasteurianum, and a Bacillus spp.

A surface antigen of Entamoeba histolytica was demonstrated to be a plasma membrane antigen by immunoprecipitation of metabolically 35S-labeled antigen from live trophozoites, Triton X-114 detergent extracts, and plasma membrane-enriched fractions prepared by concanavalin A membrane stabilization and differential centrifugation. In addition, the antigen was localized to the plasma membrane by electron microscopy with colloidal gold. Antigen from E. histolytica strains immunoprecipitated with specific immunoglobulin M (IgM) or IgG2b monoclonal antibody was identical by one-dimensional peptide mapping with N-chlorosuccinimide. Additionally, antigen from different axenically cultivated amebae was demonstrated to be identical by N-chlorosuccinimide peptide mapping, as were peptide maps of IgG and IgM monoclonal antibody-purified antigen. The 96-kilodalton (kDa) surface antigen was identified on four axenically cultivated pathogenic isolates and on three polyxenically cultivated pathogenic isolates (zymodeme II) of E. histolytica but was absent or present in lesser quantity on six nonpathogenic polyxenically cultivated isolates. The 96-kDa antigen was detected in liver abscess fluid from four patients with amebic abscesses by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Two-dimensional gel electrophoresis profiles of the 96-kDa antigen purified from abscess material or from polyxenically cultivated trophozoites demonstrated that the antigens were related to the 96-kDa antigen found in axenically cultivated organisms.

Campylobacter cinaedi" was isolated from the blood of a 29-year-old homosexual man with previously diagnosed acquired immune deficiency syndrome. Subculturing of the organism was achieved with the use of 7% lysed horse blood and 10% sheep blood agars at 37°C in a microaerophilic atmosphere. Problems associated with the culturing of this organism are reviewed.

To further characterize the 29-kDa surface antigen of Entamoeba histolytica, we analyzed the complete nucleotide sequence and compared the immunoreactivity of this antigen in pathogenic and nonpathogenic strains. Five cDNA clones (one 1.0-kb full-length clone, designated p13, and four partial-length clones) encoding the antigen were analyzed for allelic variation. Comparison of the nucleotide sequences revealed several single-nucleotide substitutions in all five cDNAs, two of which resulted in amino acid differences. Localization of the antigen to the amebic surface in a previous report (B. E. Torian, B. M. Flores, V. L. Stroeher, F. S. Hagen, and W. E. Stamm, Proc. Natl. Acad. Sci. USA 87:6358-6362, 1990) was corroborated by transmission electron microscopy showing colloidal gold particles on the surface of the trophozoites. Computer analysis of the deduced amino acid sequence predicted that the protein encoded by p13 was a hydrophilic peripheral membrane protein, and these data were confirmed by a Triton X-114 membrane extraction showing the presence of the 29-kDa antigen primarily in the aqueous phase of the detergent partition. Monoclonal antibodies to a fusion peptide differentiated between pathogenic and nonpathogenic clinical strains of E. histolytica in immunoblots. Although no immunoreactive epitopes were detected on nonpathogenic strains, Northern (RNA) analysis and DNA-DNA hybridization with a 700-bp cDNA probe demonstrated that mRNA and the gene encoding the 29-kDa surface antigen were present in both pathogenic and nonpathogenic clinical isolates.