The inner body of Loa loa microfilariae. Morphologic and physiopathologic data

The innenk?rper is visible in 80% of the cases of microfilariae Loa loa we have studied and stained at pH 7.2. In thick blood film, however, it was not visible. It lies between the middle of the body and the G1 = R1, cell. The central viscus rarely take the form of a single elongated mass, it most often appears moniliform with 2 to 5 granular masses and it is sometimes reduced to a few number of granulations. Its average length is 34 microns. As with W. bancrofti it would appear to have the role of a nutritional reserve.     

Cloning and characterization of a species-specific repetitive DNA sequence from Loa loa

A genomic DNA library of Loa loa was constructed in lambda gt11 using EcoRI-digested DNA from microfilariae isolated from two West African patients. Screening with labeled L. loa DNA yielded several potential repetitive DNA clones. An MboI fragment of one of these, LL3M9, was identified and characterized. Sequence analysis of LL3M9 revealed an 839-bp fragment with an unusual 356-bp region containing 37 copies of the hexamer CTTAGG, many of which are arranged in repeated motifs of 12, 27 and 63 bp. This region shares many of the characteristics of eukaryotic satellite DNA. A synthetic oligonucleotide corresponding to the 27-bp repeated motif, LL3M9REP, was found to be both sensitive and species-specific by dot hybridization. Species specificity of LL3M9REP was confirmed by amplification of the repetitive region using genomic DNA as a template in the polymerase chain reaction.     

Loa loa filariasis: cause of severe eosinophilia

Two cases of very high hypereosinophilia (28,160 and 11,232/mm3) observed in congolese patients are presented. Although microfilaraemia was not detectable, loiasis was diagnosed, given the clinical manifestations, epidemiological data, history of sub-conjunctival migration of the adult worm (in one case), spectacular recovery (clinical and biological) after treatment with diethylcarbamazine. This "allergic form" of filariasis is often considered unusual in indigenous subjects.     

The identification and partial characterization of an immunodominant 29-31 kilodalton surface antigen expressed by adult worms of the human filaria Loa loa

Detergent solubilized extracts of 125Iodogen surface labelled adult Loa loa revealed a relatively simple profile consisting of a strongly labelled molecule at 29-31 kDa and weakly labelled molecules at 14.5, 17, 21, 23, 34, 58, and 86 kDa. Residents of a L. loa endemic zone were assessed clinically and parasitologically and classified as microfilaremic, amicrofilaremic with documented ocular passage of adult worms, or 'resistant' subjects without any signs of infection. Sera from these subjects were used to identify L. loa adult surface antigens. All 'resistant' sera immunoprecipitated the 29-31 kDa antigen although some were more strongly reactive than others. The amicrofilaremic sera strongly immunoprecipitated the 29-31 kDa antigen, whereas microfilaremic sera reacted weakly or not at all with this antigen. Longer exposures of immunoprecipitates of strongly reactive sera revealed the recognition of additional antigens of 86, 44, 34, 23, 21, 17 and 14.5 kDa. Studies with heterologous sera demonstrated that these antigens contain cross-reactive epitopes which are restricted to filarial parasites. Biochemical characterization of the predominant 29-31 kDa antigen showed that it bound concanavalin A, was sensitive to proteases, and its antigenicity was resistant to heat but sensitive to periodate and endo-beta-N-acetylglucosaminidase H. These observations suggest that it is a glycoprotein containing mannose and N-acetylglucosamine residues and that the carbohydrate moiety is important for antibody binding. The importance of the 29-31 kDa glycoprotein in the immunobiology of loaiasis is suggested by the finding that resistant and infected amicrofilaremic individuals have strongly reactive IgG antibodies to this antigen.     

Immunocytochemical localization of surface antigens in microfilariae of Wuchereria bancrofti

Antigenic sites were localized on the surface structure (sheath and cuticle) of microfilariae of Wuchereria bancrofti using sera from microfilaraemic and amicrofilaraemic patients using antibodies associated to colloidal gold particles and transmission electron microscopy. No labeling was observed on the surface of intact parasites. Microfilariae without the sheath and isolated sheath were obtained by ultrasound treatment. Using these preparations labeling of the inner portion of the sheath was observed when sera from amicrofilaraemic patients were used. Labeling of the cuticle surface was observed mainly in the regions of the cuticular annulations.     

Loop-Mediated Isothermal Amplification for Rapid and Semiquantitative Detection of Loa loa Infection

Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic.     

Biochemical characterization of tumor-specific cell surface antigens on avian oncornavirus transformed cells.

A sensitive indirect immune precipitation method coupled with polyacrylamide-gel electrophoresis was used to detect new antigens from avian oncornavirus-transformed chicken embryo cells (CEC). A total of four antigens were recognized by this method. Two of these antigens appeared to be identical to the gp85 and gp37 components of the virus envelope. The other two antigens were distinct from the viral envelope antigens on the basis of both size and antigenicity, and were group specific for the avian oncornavirus system. The larger nonvirion antigen was detectable on transformed CEC but absent from normal or productively infected nontransformed CEC and therefore was clearly tumor specific. Because of the weak detectability of the smaller nonvirion antigen, its tumor specificity could not be decided. The tumor-specific antigen was a glycoprotein of 100,000-dalton apparent molecular weight, whereas the smaller nonvirion antigen was 32,000 daltons and probably also a glycoprotein. The 100,000-dalton tumor-specific antigen was found on the cell surface and therefore represented a tumor-specific cell surface antigen (TSSA).     

Enzyme profile and immunochemical characterization of Aspergillus fumigatus antigens

We have compared the immunochemical characteristics of culture-filtrate antigens (Ag) from Aspergillus fumigatus extracted in our laboratory with commercially available Ags. A total of 20 different preparations were studied for protein and carbohydrate content, presence of endotoxins, mycotoxins, and hemolytic toxins. These extracts were analyzed by two-dimensional electrophoresis for protein components. The immunogenicity of the preparations was determined by rocket electrophoresis with rabbit anti-A. fumigatus sera and by agar gel diffusion with sera from patients with allergic bronchopulmonary aspergillosis, aspergilloma, and normal control subjects. In order to have dependable immunologic results, the Ags must be sufficiently pure and reproducible. Until such time as pure and standardized Ags are available, the crude Ags used should be characterized to the extent that adequate reproducibility between preparations can be ascertained. The enzyme profile of the Ag preparations provides a fair indication of the quality of antigenic components, and together with other immunochemical parameters, it will be of use in determining the suitability of the extracts in immunodiagnosis. Immunochemical results demonstrate that commercial Ags contain less proteins and carbohydrates and fewer enzymes than the homemade antigens. In addition, fewer patients demonstrated specific precipitins against commercial Ags than with homemade Ags. This study once again confirms the need for pure standardized Ags for studying the immunologic response in patients with Aspergillus-induced diseases. Until such preparations are readily available, partially purified or crude Ags with known immunochemical properties and enzyme profile may be the choice for immunodiagnosis.     

Immunochemical characterization and biosynthesis of major antigens of iodo-bead surface-labeled Brugia malayi microfilariae

The objectives of the present study were to identify and characterize biochemically the major antigens of Brugia malayi microfilariae, a filarial parasite that infects humans. IgG antibodies in sera of mice which had cleared parasites from the bloodstream reacted with 30, 55, 94 and 150 kDa molecules of living microfilariae radioiodinated by the Iodo-bead method. Sera of humans infected with the related filariae Wuchereria bancrofti, Loa loa or Onchocerca volvulus immunoprecipitated molecules of similar size as well as two additional proteins of 22 and 43 kDa. Sera of uninfected North Americans or mice infected with Trichinella spiralis or Schistosoma mansoni did not recognize these radioiodinated antigens. Experiments to examine the possible surface localization and metabolism of these antigens showed that they were removed from intact parasites exposed to chymotrypsin or trypsin and that immunogenic molecules of 30, 55, and 150 kDa were released into excretory-secretory products by viable microfilariae. [35S]Methionine biosynthetically labeled polypeptide antigens of 22, 30, 35 and 150 kDa were detected by antibody reacted with intact microfilariae and/or their excretion products. Antigens of 30, 55, and 150 kDa appear to be glycoproteins as they bound wheat germ agglutinin and were biosynthetically labeled with [14C]N-acetyl-D-glucosamine. These data suggest that the surface of B. malayi microfilariae is a dynamic structure which synthesizes and sheds antigens.     

Biochemical characterization of avian T lymphocyte-specific antigens

Sera from rabbits injected with quail thymocytes were absorbed on quail bursal and liver cells. The absorbed sera reacted with avian T but not B lymphocytes in an immunofluorescence assay. Material precipitated by the anti-T antisera from lysates of radioiodinated chicken or quail thymocytes was analyzed by one- and two-dimensional electrophoresis. Of two anti-T cell antisera studied in detail, one reacted mainly with a protein of an apparent molecular weight of 45 000 - 55 000, and the other with a protein of an apparent molecular weight 65 000 - 70 000. These proteins may be homologues of previously described mammalian T lymphocytes-specific antigens.     

Biochemical and immunological characterization of major surface antigens ofSarcocystis muris andS. suicanis cyst merozoites

Surface proteins ofSarcocystis cyst merozoites were labeled by biotinylation or radioiodination and identified on Western blots after sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The major labeled proteins ofS. muris andS. suicanis have relative molecular masses of 31 and 33 kDa, respectively. Immunoblots performed with the 31-kDa protein and sera of experimentally infected mice or with monoclonal antibodies toS. muris revealed that this protein is immunogenic. Indirect fluorescent antibody tests (IFAT) executed usingSarcocystis cyst merozoites and polyclonal monospecific antibodies obtained from rabbits immunized with homogeneous major surface antigens gave additional evidence for the localization of the identified antigens in the pellicle. Analysis ofS. muris andS. suicanis proteins by two-dimensional gel electrophoresis revealed multiple isoelectric forms.Supported by the Deutsche Forschungsgemeinschaft and dedicated to Prof. Dr. Johannes Eckert (Zürich) on the occasion of his 60th birthday     

Biochemical and immunochemical characterization of 125I-labeled cuticle components of Haemonchus contortus

Live Haemonchus contortus developmental stages were radioiodinated and then subjected to a stepwise extraction procedure consisting of a buffer extract (with or without detergent) to solubilize putative surface-associated antigenic macromolecules, followed by a detergent/beta-mercaptoethanol (BME) extract to solubilize putative cuticle collagen proteins. A buffer-extracted iodinated 100-kDa protein was present in the free-living, infective L3(2M) stage. This labeled protein was released during in vitro exsheathment of L3(2M) and was not present in the ecdysed second molt (2M) cuticle. In addition to the 100-kDa protein, exsheathment fluid contained a 70-kDa labeled protein that was not extracted from iodinated L3(2M) with either detergent or BME. The data suggest that these proteins are components of the specialized ring portion of the 2M cuticle that is enzymatically ruptured during ecdysis. The L3(2M) and the exsheathed third-stage larvae (L3) contained 3 labeled, BME-extracted, collagenase-sensitive proteins of 108, 88 and 53 kDa. In contrast, four detergent-extracted, collagenase-insensitive, iodinated proteins (143, 81, 58 and 30 kDa) were present in adult H. contortus. The 143-kDa protein was both glycosylated and immunogenic. All 4 adult cuticle proteins were released from the cuticle surface into culture fluids. Furthermore, a cysteine protease was secreted by adults which apparently hydrolyzed the released 81-, 58- and 30-kDa surface proteins.     

Transmission intensity affects both antigen-specific and nonspecific T-cell proliferative responses in Loa loa infection

T-cell proliferative responses were studied in two villages in Gabon with different levels of Loa loa transmission. The first village (Okoumbi) had an annual transmission potential (ATP) of approximately 9,000 infective larvae (L3)/person/year (high transmission village), while the second village (Ndjokaye) had an ATP of approximately 1,000 L3/person/year (low transmission village). Proliferation and cytokine assays were performed on peripheral blood mononuclear cells (PBMC) from individuals aged 18 years and over using either mitogens (concanavalin A or phytohemagglutinin), antigens (purified protein derivative [PPD], irrelevant antigen), or soluble extracts of L3, microfilariae, or adult L. loa. PBMC from individuals in the low transmission village responded better to stimulation with adult antigen and to PPD than did PBMC from individuals in the high transmission village (P = 0.0031 and P = 0.0012, respectively). These data suggest that high levels of transmission of L. loa depress both specific and nonspecific T-cell proliferative responses in infected humans.     

Loa loa in the anterior chamber of the eye: a case report

An unusual case of loiasis from Assam is reported here. Loa loa is a subcutaneous filarial parasite of man and is transmitted to humans by chrysops flies. The patient presented with foreign body sensation and visual disturbances of the right eye. Examination revealed a white coiled structure in the cornea. Routine blood and other investigations were within normal limits. A live adult worm was extracted and identity was confirmed by microscopy to be Loa loa. Patient was treated with diethylcarbamazine and steroid. We found this case interesting as the worm was present in the anterior chamber--an unusual site and there were no other positive findings besides the lone worm.     

Biochemical and immunological characterization of soluble antigens of Giardia lamblia

The crude soluble antigens (CSA) of Giardia lamblia trophozoites and their analytically purified fractions were characterized biochemically and immunologically to determine the most immunogenic fraction and its localization on the parasite. Both Sephacryl S-300 column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the highly complex and heterogeneous nature of CSA. Gel filtration of CSA showed four fractions (FI–FIV) with molecular masses of 250, 150, 110, and 10 kDa for fractions I–IV, respectively. Protein profiles of CSA demonstrated 28 Coomassie-blue bands in the range of 200–14 kDa. Similar banding patterns with fewer polypeptides were observed in the FI fraction. However, fractions II and III showed polypeptide bands in the region of 97–14 kDa. The glycoprotein nature of CSA and its fractions were demonstrated in physicochemical analysis. In antigenic activity analysis the high-molecular-weight FI antigen was found to be 8 times more immunogenic than CSA as well as the other fractions. Major differences in the immunoreactivity of CSA and FI antigens were noted at 220, 30, and 22 kDa for the FI antigen and at 205, 84, 55, 43, and 20 kDa for CSA. Some of these FI polypeptides were found to be surface-associated as revealed by immunofluorescence and immunoblot assay. These results suggest the future use of the FI antigen in the serodiagnosis of and immunoprophylaxis against giardiasis. Received: 20 September 1996 / Accepted: 29 November 1996