The first case of tsutsugamushi disease in Ehime Prefecture

The first case of tsutsugamushi disease in Ehime Prefecture was experienced in December 1987 with successful isolation of the causative agent. The patient was taken ill twelve days after infection. Immunofluorescent antibody tests using the isolate, Yamazaki strain, and Gilliam, Karp, Kato, Irie and Shimokoshi strains as antigens revealed that the specific antibodies against these antigens appeared and increased in the blood of the patient during the course of the disease. And the antibody titers to the Yamazaki antigen were the highest of these antigens. Agglutinin for Proteus OXK did not appear in the blood of the patient. The immunofluorescent antibody test using type-specific monoclonal antibodies to Gilliam, Karp, Kato, Irie and Shimokoshi strains and these five strains and the Yamazaki strain as antigens revealed that the Yamazaki strain was identified as Karp type of Rickettsia tsutsugamushi.     

Clinical and etiological studies of tsutsugamushi disease in Miyazaki district--correlation of serological type of R. tsutsugamushi to clinical feature

The correlation of pathogenic and immunologic characteristics of Rickettsia tsutsugamushi to clinical findings of patients with tsutsugamushi disease in Miyazaki was investigated. In two immunological types, Hirano type strains seemed to have higher virulence to mice than Irie from the findings during the course of infection and on autopsy. A strain of Hirano type was so virulent as to succumb to the infection. As to clinical findings, incidence of hepatomegaly was slightly higher in Hirano type patients than Irie, which is one of the signs in severe type tsutsugamushi disease. This was supported by the higher mean value and frequent appearance of abnormality in liver function test, sGOT, sGPT and LDH, in this type of patients.     

Antigenic types of Rickettsia tsutsugamushi isolated from patients with tsutsugamushi fever and their distribution in Miyazaki Prefecture

Rickettsia tsutsugamushi (Rt) isolated from patients with tsutsugamushi fever were examined for their antigenicity. This was done by indirect immunofluorescence (IIF) with guinea pig antisera against three standard strains (Karp, Kato and Gilliam) and two local strains (Kawasaki and Kuroki) isolated in 1981, and with mouse monoclonal antibodies against the three standard strains. In the meantime, antibodies in sera from 317 out of 442 patients registered during 1985 to 1988 were titrated by IIF with those five Rt strains. 1) Local isolates, Kawasaki and Kuroki strains, reacted most effectively with the homologous antiserum, respectively, showing four fold lower IIF titers against the heterologous antisera. 2) Kawasaki strain reacted with none of the monoclonal antibodies, whereas Kuroki strain showed a slight reaction with anti-Karp and anti-Kato, but not anti-Gilliam, monoclonal antibodies. 3) Seventeen out of 27 strains isolated in 1985 resembled the Kawasaki strain in their reaction patterns with the antisera and monoclonal antibodies, and the other 10 strains showed reactivity similar to the Kuroki strain. 4) Sera of 233 (74%) out of 317 patients showed the highest antibody titers against the Kawasaki strain and 69 (22%) of 317 against the Kuroki strain. It is thus evident that Kawasaki and Kuroki strains are antigenically different from the standard strains, and Kawasaki and Kuroki strains also differ from each other. It is suggested that two antigenic types (Kawasaki and Kuroki) of Rt were distributed in Miyazaki Prefecture, Rt of the Kawasaki type slightly dominates Rt of the Kuroki type, and recent tsutsugamushi fever has been caused by either one or the other type of Rt.     

Studies on tsutsugamushi diseases in Gifu Prefecture. 5. Characterization of monoclonal antibodies to prototype strains of Rickettsia tsutsugamushi and immunological grouping of newly isolated strains using the antibodies]

We characterized 8 monoclonal antibodies (MAbs) to Karp, Kato, and Gilliam strains of Rickettsia tsutsugamushi, and analysed 17 isolates from patients with Tsutsugamushi disease using these MAbs. These were divided into 3 strain-specific (Kp/D11, Kt/2D9, and Gi/E4) and 5 cross-reactive MAbs (Kp/1F11, Kp/1C10, Kp/C6, Kt/3B2, and Kt/3C2). All MAbs recognized characteristic protein antigens using the indirect fluorescent-antibody test (IFA) and proteinase K treatment. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that Kato-specific MAb Kt/2D9 recognized a polypeptide with a molecular mass of 54 kilodalton (kDa) of the homologous strain, and cross-reactive MAbs Kp/1F11, Kp/C6, and Kt/3B2 recognized those of 46-47 kDa, 46-47 KDa, and 60 kDa, respectively to the homologous and heterologous strains. MAbs Kp/1C10 which exhibited a high IFA titer against the Karp strain and only low titers against heterologous strains recognized only the 110 kDa polypeptide of the homologous strain. MAb Kt/3C2 which reacted with both Karp and Kato strains recognized a 54 to 56 kDa polypeptide band of the two prototype strains as well as several other polypeptides, however, each molecular mass was present in only one of two strains. Testing by the plaque reduction technique showed another characteristic of MAb Kt/3C2 to neutralize both Karp and Kato Strains. Fourteen isolated strains from patients in the south and west regions of Gifu Prefecture, the Shimokoshi stain isolated in Niigata Prefecture, and Kawasaki and Kuroki stains isolated in Miyazaki Prefecture were examined for reactivities to 8 MAbs by IFA to classify their antigenicities. No isolated strains reacted with Karp-specific Kp/D11, Kato-specific Kt/2D9, or Gilliam-specific Gi/E4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and use of monoclonal antibodies for identifying Crimean-Congo hemorrhagic fever virus

Seven monoclonal antibodies were prepared against a South African strain of Crimean-Congo hemorrhagic fever (CCHF) virus and were found to be directed against viral nucleocapsid protein. Five of the monoclonal antibodies reacted to high titer in indirect immunofluorescence (IF) tests and enzyme-linked immunosorbent assays (ELISA) with 22 strains of CCHF virus and failed to cross-react with the closest antigenic relative of CCHF, Hazara virus, or with 4 other nairoviruses which need to be distinguished from CCHF virus in Africa. These antibodies, used in the IF technique, readily detected antigens induced by all strains of CCHF virus included in the study in cell culture monolayers and mouse brain tissue, which represent the systems commonly used for isolation of CCHF virus. The IF technique with monoclonal antibodies constitutes a rapid and specific means of identifying newly isolated strains of CCHF virus.     

Serological and genetic characterization of bovine rotaviruses in Thailand by ELISA and RNA-RNA hybridization: detection of numerous non-serotype 6 strains

A total of 62 fecal specimens positive for rotavirus were collected from diarrheic cows in Thailand in 1988 and 1989. The antigenic properties of rotaviruses in stool were examined by enzyme-liked immunosorbent assays using specific monoclonal antibodies directed at VP4, VP6 or VP7: all the bovine rotavirus strains were determined as subgroup I; none of the strains were reactive with serotype 6-specific monoclonal antibody; and different reactivities of the bovine strains with two anti-VP4 monoclonal antibodies were observed. Polyacrylamide gel electrophoresis of viral RNA exhibited three different RNA electropherotypes. In RNA-RNA hybridization experiments using cell culture-adapted three strains as well as a reference bovine strain (NCDV), RNA from the Thai bovine strains showed very low homology to that from NCDV; only three or four RNA segments were hybridized between the RNAs from Thai samples and NCDV. These results suggested that bovine rotaviruses isolated in Thailand are serologically and genetically distinct from a reference serotype 6 bovine strain, NCDV.     

Variations in the replication and antigenic properties of human herpesvirus 6 strains

The Z29 and U1102 strains of human herpesvirus 6 (HHV-6) were compared for their ability to replicate in fresh peripheral blood lymphocytes (PBL) and in continuous T cell lines. The replication of both strains in PBL was enhanced by mitogenic activation of cell growth. U1102 replicated in the continuous T cell lines, J JHAN and HSB-2, whereas no Z29 replication was observed in these cell lines as judged by infectious virus yields, the presence of viral antigens, and viral DNA replication. The two strains were compared with respect to their ability to react in immunofluorescence assays with monoclonal antibodies (MAbs) prepared against the GS strain of HHV-6. These MAbs are directed against six different polypeptides including three glycoproteins. All MAbs reacted with cells infected with the U1102 strain. The Z29-infected cells reacted with four MAbs but failed to react with MAbs specific for an 82- to 105-kDa major surface glycoprotein and with one MAb reactive with a nonglycosylated 180-kDa protein. Taken together, the two strains of HHV-6 exhibit variations with regard to their growth and antigenic properties.     

Genes for the major protein antigens of Mycobacterium tuberculosis: the etiologic agents of tuberculosis and leprosy share an immunodominant antigen.

Mycobacterium tuberculosis genes encoding immunologically relevant proteins were isolated by systematically screening a lambda gt11 recombinant DNA expression library with a collection of murine monoclonal antibodies directed against protein antigens of this pathogen. These antibodies, previously characterized by a World Health Organization workshop on monoclonal antibodies against mycobacteria, were used to isolate DNA sequences encoding five major protein antigens of this pathogen. To evaluate the extent of crossreactivity between these M. tuberculosis antigens and antigens of Mycobacterium leprae, recombinant antigens were probed with monoclonal antibodies directed against the protein antigens of these bacilli. One of the antigens, a 65-kDa protein, has determinants common to M. tuberculosis and M. leprae. We find not only that this antigen is recognized by mouse monoclonal antibodies but that it is the major protein recognized by anti-M. tuberculosis rabbit sera. The 65-kDa proteins of M. tuberculosis and M. leprae appear to play a role in the humoral and cell-mediated immune response to these pathogens.     

Monoclonal antibodies against plasma protease inhibitors: production and characterization of 15 monoclonal antibodies against human antithrombin III. Relation between antigenic determinants and functional sites of antithrombin III

Fifteen hybridomas secreting monoclonal antibodies against human antithrombin III, originating from two mouse strains, have been produced by the cell fusion technique. Eight monoclonal antibodies belong to the class IgG1, five to the class IgG2a, and two to the class IgG2b. All light chains belong to the kappa group. No cross-reaction of the monoclonal antibodies have been observed with a crude preparation of albumin nor with alpha 1-antitrypsin and alpha 2-antiplasmin. Five of these monoclonal antibodies exhibit a relatively high avidity for antithrombin III. Inhibition experiments showed that the 15 monoclonal antibodies define seven more or less independent antigenic regions on the antithrombin III molecule. Examination of the effects of these antibodies on the inhibitory capacity of antithrombin III toward thrombin activity, either in the presence or in the absence of heparin, showed that several monoclonal antibodies inhibit the antithrombin III activity and allowed to relate some of the antigenic determinants to functional sites on the antithrombin III molecule.     

Case Report: Tsutsugamushi Disease Caused by Shimokoshi-Type Orientia tsutsugamushi: The First Report in Western Japan

An 85-year-old female farmer was admitted to our hospital for fever, general fatigue, and skin rash. Cephalosporin was not effective and minocycline was dramatically effective. An eschar was discovered on her inguinal region after the defervescence. Laboratory examination of serum taken 12 days after onset of the illness showed elevated titers of antibodies against the Shimokoshi strain of Orientia tsutsugamushi. The gene sequence analysis of specimen from the patient''s eschar revealed high similarity to the Shimokoshi strain by nested polymerase chain reaction. Therefore, this patient was diagnosed as a case of Shimokoshi-type tsutsugamushi disease, which has not previously been reported in Western Japan. Recently, cases of this type have also been confirmed in northeastern Japan, suggesting the need for further epidemiological studies.     

Analysis of prevalent Orientia tsutsugamushi in Aichi Prefecture

Orientia tsutsugamushi was isolated from one of 8 patients' sera in Aichi Prefecture, and was identified to have the same antigenicity with the KN-2 strain (KN-2 like) based on the reactivity with 13 types of strain-specific or cross-reactive monoclonal antibodies to Karp, Gilliam, and Kato strains. Four isolates from 4 unfed larvae and adult of Leptotrombidium pallidum were also classified as the KN-3 like strains. Using indirect immunofluorescence, sera from 20 patients with tsutsugamushi disease were tested for reactivity with KN-1, KN-2, KN-3, and GJ-1 strains, isolated from patients in Gifu Prefecture. Fifteen sera showed the highest titer against KN-2 strain in Immunogloburin M (IgM). Of the other 5, three were higher for KN-3 strain in IgM, and two were KN-1 or GJ-1, respectively. These results suggested that KN-2 like strains were prevalent in the region where the number of patients has been ranked the highest in Aichi Prefecture. KN-1, KN-3, and GJ-1 like strains were also existed in this area. KN-3 like strain was likely to be distributed in another area. Aichi Prefecture.     

Susceptibility of phenotypic variants of Haemophilus influenzae type b to serum bactericidal activity: relation to surface lipopolysaccharide

After three serial passages of Haemophilus influenzae type b strain Fuju in rats, we recovered a variant, which differed in colonial morphology, serum sensitivity, and lipopolysaccharide configuration from the parent strain. The parent organism (Fuju) appeared iridescent and transparent on Levinthal agar, and the rat-passaged variant (rat3 Fuju) appeared iridescent and opaque. The transparent, parent strain Fuju was sensitive to the complement-mediated bactericidal activity of normal rat serum, and the opaque, rat-passaged strain rat3 Fuju was serum resistant. Serum killing of the serum-sensitive strain appeared to be mediated by the classic complement pathway. Both serum-resistant and serum-sensitive strains were killed equally well by immune rat and human serum. These two strains did not differ in the amount of capsular polysaccharide that they elaborated nor in their major outer membrane protein patterns on SDS-PAGE. Lipopolysaccharide isolated from these two strains demonstrated different electrophoretic mobility patterns. Furthermore, the organisms showed different reactivities with two monoclonal antibodies directed against determinants of Hib lipopolysaccharide. Thus, the difference in susceptibility to complement-mediated bactericidal activity of normal rat serum displayed by these two strains is associated with their phenotypes, appears to be unrelated to differences in major outer membrane proteins or in the amounts of capsular polysaccharide elaborated, and is associated with differences in surface lipopolysaccharides.     

Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies

The relative binding sites of dengue serotype-specific, dengue subcomplex-specific, dengue complex-specific, flavivirus subgroup-reactive, and flavivirus group-reactive monoclonal antibody preparations were identified by using competitive antibody binding assays. A dengue complex-specific epitope, capable of mediating infection enhancement, was identified on a 20,000 dalton protein found on intracellular virions. The other epitopes were assigned relative positions on the E glycoprotein by competitive antibody binding. These could be grouped into 3 linkage groups based on the ability of some monoclonal antibodies to block contiguous binding sites. Some antibodies were able to increase or "promote" the binding of antibodies from other linkage groups. These results suggest that a continuum of antigenic reactivities exist on the E glycoprotein of the dengue viruses, and that the conformation of this glycoprotein may be altered after antibody binding.     

Monoclonal antibodies specific for Hantaan virus.

Six hybridoma cell line producing monoclonal antibodies to Hantaan virus were established by fusion of NS-1 mouse myeloma cells with spleen cells of mice immunized with Hantaan virus strain 76-118. The specificity of these monoclonal antibodies was established by immunoblotting analysis and immunofluorescence. Five of the clones reacted with antigens on the cell surface and in the cytoplasm, and one clone reacted with a determinant expressed only in the cytoplasm of the infected cells. Two of the clones produced antibodies that reacted with a Mr 50,000 polypeptide in virus-infected cellular extracts and purified virus preparations. The monoclonal antibodies were used to examine the antigenic relationship among Hantaan virus strains and between Hantaan virus and Prospect Hill virus and the virus of nephropathia epidemica. Three antibodies were capable of distinguishing between the Lee strain and the 760-118 strain of Hantaan virus and three additional antibodies reacted with determinants shared by both virus strains. None of the six reacted with Prospect Hill virus or the virus of nephropathia epidemica.     

Characterization of monoclonal antibodies against Schistosoma mansoni

Monoclonal antibodies directed against Schistosoma mansoni antigens were produced by the in vitro fusion of B lymphocytes, obtained from mice infected with S. mansoni, and SP2/0 myeloma cells. Antibody reactivity was assessed by ELISA binding, utilizing 4 M KCl extracts of cercariae and adult worms, soluble egg antigen (SEA), and purified antigenic preparations, and by indirect immunofluorescence using living schistosomula. The monoclonal antibodies recognized a wide spectrum of antigenic determinants. The specificity of the monoclonal reactivities ranged from high cross-reactivity to extreme restriction, vis-a-vis the distribution of the recognized determinants within genus, species, stages, and purified antigenic preparations. The specificity of reactivity of monoclonal antibodies for a given determinant was greater than that of immune mouse serum. These studies establish the feasibility of the production of large numbers of monoclonal antibodies and of their use of antigen identification. The monoclonal antibodies are available to interested investigators upon request.     

Geographic variation among isolates of Trichomonas vaginalis: demonstration of antigenic heterogeneity by using monoclonal antibodies and the indirect immunofluorescence technique

Although Trichomonas vaginalis causes one of the most common sexually transmitted diseases, little is known about the antigenic variation of the parasite or about differences between strains in epidemiology or virulence. Variation among isolates of T. vaginalis was investigated by using a panel of monoclonal antibodies, each reactive with different antigens, to test 88 isolates from diverse geographic areas of North America. All isolates of T. vaginalis reacted with at least one of the nine monoclonal antibodies; the individual antibodies reacted with 22%-76% of the isolates. A pool of two broadly reactive antibodies identified all isolates in the study. Four of the most narrowly reactive, or "specific," antibodies demonstrated differences in the antigenic composition of trichomonads isolated from patients in Seattle, Baltimore, and Brooklyn, New York (P less than .005 by chi 2 test). Application of these and other monoclonal antibody probes may facilitate epidemiological studies and provide rapid, reliable methods for direct diagnosis of trichomonads in clinical specimens.     

Identification of strain-specific and group-reactive antigenic determinants on the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi

Hybridoma antibodies (Hab) were prepared against the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi and were examined for homologous and heterologous reactivity using an indirect immunofluorescence assay. Strain-specific Hab demonstrated homologous IFA titers ranging from 1/320 to 1/1,280 and did not react (less than 1/10) with the heterologous strains. The cross-reactive Hab generally reacted equally with all three strains in the scrub typhus group; however, there were some Hab that reacted with only one of the two heterologous strains tested. The Hab also were examined in enzyme-linked immunosorbent assays with scrub typhus antigens eluted from SDS-polyacrylamide gels. Most Hab reacted with either one or several of the six eluted antigens detected with a polyclonal immune serum. It was also observed that strain-specific and cross-reactive Hab sometimes reacted with the same antigen, suggesting the existence of multiple antigenic determinants in one electrophoretic peak. The data suggest that strain-specific Hab can be used in the indirect immunofluorescence assay to identify isolates of R. tsutsugamushi without the cross-reactions usually observed with polyclonal antisera, and that they are useful probes for detection and analysis of rickettsial antigens.     

Monoclonal antibodies to metacyclic stage antigens of Trypanosoma cruzi

Hybridoma cell lines secreting monoclonal antibodies against the Tulahuén strain of Trypanosoma cruzi were produced by the fusion of SP2/O-Ag 14 myeloma cells with spleen cells from mice immunized with irradiated metacyclic trypomastigotes. Twenty of the monoclonals synthesized by the hybridomas were identified as IgM, 2 as IgG1, 10 as IgG2a, 3 as IgG2b, 4 as IgG3, 1 as IgE, and 1 as IgA. Twenty-three of these antibodies had kappa light chains and 18 showed delta chains. Twelve of the monoclonals agglutinated metacyclic trypomastigotes without additional concentration and 4 of these precipitated antigens in extracts of T. cruzi metacyclic or epimastigote stages. One monoclonal precipitated an epimastigote antigen, while another reacted with a metacyclic antigen, and 2 antibodies formed precipitin lines with antigens of both stages. Agglutinin assays performed to characterize surface antigenic specificities of the 12 monoclonal antibodies showed that 2 reacted only with the metacyclic stage of the Tulahuén strain. Two monoclonals agglutinated both metacyclic trypomastigotes and epimastigotes of the Tulahuén strains. Three antibodies caused clumping of metacyclics and epimastigotes of the Tulahuén, Raccoon V, and Corpus Christi strains of T. cruzi, while a fourth also reacted with bloodstream trypomastigotes. One monoclonal detected identical epitopes on metacyclics and epimastigotes of T. cruzi and epimastigotes of Trypanosoma musculi, while 2 antibodies reacted with metacyclics, epimastigotes, and bloodstream trypomastigotes of the Tulahuén and Raccoon V strains and the bloodstream stage of T. musculi. One antibody agglutinated all stages and strains of T. cruzi, T. musculi, and Trypanosoma lewisi which were tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variable antigen type (VAT) composition of Trypanosoma brucei rhodesiense: discrepancy between results obtained using VAT-specific monoclonal antibodies and rabbit antisera

Monoclonal antibodies have been made against clones of Trypanosoma brucei rhodesiense from the WRATAR 1 serodeme and analyzed by indirect immunofluorescence assay for specificity against homologous and heterologous clones. These antibodies were shown to be variable antigen type (VAT)-specific as they identified the majority of parasites in the homologous clones but few parasites in other clones. The reactivities of these VAT-specific monoclonal antibodies with uncloned human trypanosome isolates from Kenya were compared with the reactivities of polyvalent, VAT-specific rabbit sera on the same isolates. Different reaction patterns were obtained with the two sets of reagents and concordant reactions were less than 20%. Our data indicate that single monoclonal antibodies are not interchangeable for sera in the typing of antigenic variants of African trypanosomes, and multiple monoclonal antibodies for each antigenic variant will probably be needed.     

Immunological analysis of Japanese encephalitis virus using anti-Kamiyama monoclonal antibodies

Fifteen hybridomas were produced by fusing P3X63Ag8.653 mouse myeloma cells with spleen cells from BALB/c mice immunized with Japanese encephalitis (JE) virus Kamiyama strain. Antigenic analysis of twenty-five strains of JE virus was carried out by hemagglutination inhibition (HI) test with anti-Kamiyama monoclonal antibodies (KAMIMAs). Twenty-one JE virus strains were isolated from various parts of Japan, and four foreign countries. These strains had been isolated from different host between 1935 and 1979. According to the HI test against the five species-specific monoclonal antibodies, the twenty-five JE virus strains were classified serologically as follows: Group A: Kamiyama, Sekiya, Mochizuki, Nishizono, Sasazaki, Mie 44-1, Fukuoka 7101, Fukuoka 7202, Fukuoka 7309, Fukuoka 7311, Fukuoka 7452, Fukuka 7463, Fukuoka 7506, Kumamoto 80679, Chang Mai and JaGAr 02 strains. Group B: Nakayama-RFVL and Nakayama-Yoken strains. Group C: Nakayama-Yakken, Kalinina, G-1 late, JaGAr 01, Beijing 1 and 691004 strains. Group D: Muar strain. These results mostly corresponded with the serological classification by anti-Nakayama-RFVL monoclonal antibodies. Analysis of the biological activities of KAMIMAs revealed that there were no correlations among HI titer, ELISA titer and neutralization titer. Neutralizing and some non-neutralizing monoclonal antibodies protected mice infected with lethal doses of JE virus Kamiyama strain. SDS-PAGE and immunoblotting analysis revealed that three antibodies reacted with a 52.0 kD band under non-reducing conditions and with a 53.0 kD band under reducing conditions, five antibodies reacted with a 52.0 kD band only under non-reducing conditions, and that seven antibodies reacted with a 14.5 kD band under both non-reducing and reducing conditions.