Immunotyping of Chlamydia psittaci by indirect immunofluorescence antibody test with monoclonal antibodies

Monoclonal antibodies against pigeon and budgerigar strains of Chlamydia psittaci were used to classify the immunotypes of C. psittaci strains by an indirect immunofluorescence antibody (IFA) test. Thirty-three C. psittaci strains from pigeons and 24 from budgerigars were divided into three immunotypes (P-I, P-II, and P-III) and (B-I, B-II, and B-III), respectively. Two strains from human psittacosis patients were identified as P-III and B-I, coinciding with the epidemiological evidence of each human infection. Two strains from psittacine birds, a parrot and a parakeet, were identical to the B-II immunotype. Other mammalian strains were quite distinct from avian strains in their IFA reaction with the monoclonal antibodies.     

Monoclonal antibodies against Chlamydia psittaci

Five monoclonal antibodies were prepared against Chlamydia (C.) psittaci strain Pigeon-1041 isolated from a feral pigeon in Sapporo. Reactions of these antibodies to chlamydiae were examined using five strains of C. psittaci and two strains of C. trachomatis in an enzyme-linked immunosorbent assay, microimmunofluorescent test and complement fixation test. The antibodies were divided into two groups: three genus-specific (A2, D2, and I21) and two strain-specific (F2 and H9) antibodies. The antigenic determinant site of A2 was KIO4 sensitive, but those of D2, F2, and H9 were not affected greatly by KIO4 treatment. Nine C. psittaci strains from feral pigeons and 16 strains from budgerigars were classified into three groups and four groups, respectively, by reaction patterns against the monoclonal antibodies.     

Immunoelectron microscopic demonstration of tissue antigens with monoclonal antibodies

Two methods of demonstrating tissue antigens by ultrastructural enzyme immunohistochemistry were tested. The monoclonal antibodies Ki-M1 and Ki-M4 were chosen for testing the methods because Ki-M1 identifies a relatively stable, and Ki-M4 a very unstable antigen. The two antibodies react selectively with human macrophages and interdigitating reticulum cells or dendritic reticulum cells of lymphoid follicles. The Ki-M1 reaction product is confined to the surface membrane. Ki-M4 reactivity is located on the surface membrane and, less often and to a lesser extent, in the cytoplasm. The technical prerequisites for reliable conservation of the antigens identified by these two antibodies were standardized. The results indicated that prior fixation in 4% paraformaldehyde is preferable for optimum preservation of stable antigens. Application of the primary antibody prior to fixation was found to be the best procedure for demonstrating unstable antigens, although nonspecific reactions were seen more often with this method.     

Immunoelectron microscopic localization of estrogen receptor with monoclonal estrophilin antibodies

The recent production of a series of monoclonal estrophilin (estrogen receptor) antibodies recognizing estrogen receptor derived from a wide variety of animals and target tissues permits the development of immunoelectron microscopic techniques for identifying estrogen receptor. We have determined suitable conditions for the ultrastructural localization of estrogen receptor in tissue sections. Localization of receptor was observed in the euchromatin, but not in the marginated heterochromatin or nucleoli of epithelial and stromal nuclei of human endometrium. Competition studies indicate that only estrogen receptor specifically inhibits nuclear staining. The absence of any specific cytoplasmic localization at the electron-microscopic level is consistent with earlier light-microscopic observations and suggests that the majority of the cellular pool of estrophilin exists in the nucleus of hormone-responsive cells.     

Arrays of hemispheric surface projections on Chlamydia psittaci and Chlamydia trachomatis observed by scanning electron microscopy.

Scanning microscopy of two strains of Chlamydia psittaci and four strains of Chlamydia trachomatis representative of the wide diversity in origin and behavior of members of the genus revealed patches of regular arrays of hemispheric projections on the surfaces of elementary bodies of all six strains. These distinctive and perhaps unique surface structure are probably present in all populations of chlamydiae.     

Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain.

The internalization of virulent Chlamydia psittaci 6BC particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously, i.e., fragile reticulate bodies (RB) appeared to be more readily phagocytized than the rigid elementary bodies (EB). Early damage of mononuclear phagocytes occurred after internalization of chlamydiae. This was followed by a decreased uptake of particles, and may explain the relatively long persistence (up to 6 h after inoculation) of free, extracellular, "swollen", and RB-like particles. Internalized particles within phagolysosomes showed varying degrees of disintegration. The subsequent influx of polymorphonuclear phagocytes and monocytes into the inflammed peritoneal cavity may explain the rapid disappearance of chlamydiae and their antigens from the peritoneal fluid. The alteration in ultrastructure of peritoneal cells and chlamydial parasites during the inflammatory process are discussed.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

Neutralization of Chlamydia psittaci with Monoclonal Antibodies

Neutralization of Chlamydia (C.) psittaci avian strain P-1041 was examined in vitro using monoclonal antibodies (MAbs). Of the 10 MAbs used, 6 were found to exhibit neutralizing capability. These include 3 against major outer membrane protein (MOMP), 1 against lipopolysaccharide (LPS) and 2 against other protein molecules [90 kilodalton (kDa) and 90/50 kDa]. Most neutralizing MAbs were dependent on complement for efficient neutralization, while a strain-specific MAb (2B5) against the 90 kDa protein displayed a different requirement for complement and neutralized the infectivity of the P-1041 at high concentrations without complement. By competitive inhibition enzyme-linked immunosorbent assay (competitive inhibition ELISA), all 3 neutralizing anti-MOMP MAbs were demonstrated to recognize different epitopes found in very close proximity to each other on the outer membrane.     

Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies.

Monoclonal antibodies directed against Uukuniemi virus glycoproteins G1 and G2 in combination with polyclonal antibodies against the nucleoprotein (N) were used to study the maturation of the virus in Golgi complexes of infected chicken embryo fibroblasts and BHK cells. Of 25 monoclonal antibodies obtained, 10 were shown to be G1 specific and 15 were shown to be G2 specific by immunoblotting and immunoprecipitation. In double-staining experiments, some of the monoclonal antibodies gave similar distributions of fluorescence as compared with the staining obtained from polyclonal rabbit anti-G1-G2 antibodies. Others, however, preferentially stained either the glycoproteins in the Golgi complex or those at the cell surface. This may indicate that the glycoproteins underwent conformational changes during their transport. Uukuniemi virus infection resulted in the vacuolization of the membranes of Golgi complexes where the maturation of the virus was taking place. Double-staining experiments with monoclonal antibodies which preferentially stained the Golgi-associated viral glycoproteins and with anti-N polyclonal rabbit antiserum showed a correlation between the progressive vacuolization of the Golgi complex and the accumulation of viral nucleoprotein in the Golgi region, suggesting that a morphological alteration of the Golgi complex may be a prerequisite for intracellular maturation of the virus. Treatment of Uukuniemi virus-infected cells with tunicamycin, a drug which inhibits N-linked glycosylation, resulted in the accumulation of both glycoproteins at an intracellular location, apparently representing the endoplasmic reticulum. Double-staining experiments showed a parallel accumulation of nucleoprotein at these sites, indicating that local accumulation of glycoproteins is required for nucleoprotein binding to intracellular membranes.     

Pyrimidine metabolism by intracellular Chlamydia psittaci.

Pyrimidine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined mutations affecting pyrimidine metabolism. C. psittaci AA Mp cannot synthesize pyrimidines de novo, as assessed by its inability to incorporate aspartic acid into nucleic acid pyrimidines. In addition, the parasite cannot take UTP, CTP, or dCTP from the host cell, nor can it salvage exogenously supplied uridine, cytidine, or deoxycytidine. The primary source of pyrimidine nucleotides is via the salvage of uracil by a uracil phosphoribosyltransferase. Uracil phosphoribosyltransferase activity was detected in crude extracts prepared from highly purified C. psittaci AA Mp reticulate bodies. The presence of CTP synthetase and ribonucleotide reductase is implicated from the incorporation of uracil into nucleic acid cytosine and deoxycytidine. Deoxyuridine was used by the parasite only after cleavage to uracil. C. psittaci AA Mp grew poorly in mutant host cell lines auxotrophic for thymidine. Furthermore, the parasite could not synthesize thymidine nucleotides de novo. C. psittaci AA Mp could take TTP directly from the host cell. In addition, the parasite could incorporate exogenous thymidine and thymine into DNA. Thymidine kinase activity and thymidine-cleaving activity were detected in C. psittaci AA Mp reticulate body extract. Thus, thymidine salvage was totally independent of other pyrimidine salvage.     

Purine metabolism by intracellular Chlamydia psittaci.

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.     

Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.     

Surface projections and internal structure of Chlamydia psittaci.

The outermost surface of the small infectious forms of Chlamydia psittaci contain geometrically arranged spikes distributed over approximately 50% of the bacterial surface. The spikes are located opposite the concave side of an electron-dense crescent-shaped chlamydial core.     

Immunoelectron microscopy in embryos

Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis.     

Detection of Chlamydia psittaci by Immunofluorescence

A direct fluorescent-antibody (FA) test was developed to detect Chlamydia psittaci in dural impressions from specimen-inoculated mice. Technical procedures for the test were compared. C. psittaci was found in mice after infection as early by the FA technique as it was by cytochemical staining methods usually used. The lymphogranuloma venereum organism was also stained by conjugated antibody to C. psittaci. A distinctive advantage of the described FA test is that organisms are identified immunologically as members of the genus Chlamydia simultaneously with their detection.