Participation of tyrosine kinase in capping, internalization, and antigen presentation through membrane immunoglobulin in BAL17 B lymphoma cells.

BAL17 cells pulsed with goat anti-IgM or anti-IgD as antigens stimulated a goat IgG specific T cell clone in terms of inositol phosphate production. The antigen-presenting capacity of BAL17 cells was inhibited by pretreatment with the tyrosine kinase inhibitors herbimycin A or genistein. Furthermore, ligand-induced capping and endocytosis of membrane immunoglobulin, monitored at the single cell level, was also blocked by herbimycin A. These results indicate that tyrosine phosphorylation plays an important role in receptor-mediated antigen presentation by B cells.

[Use of microcapsular leptospiral antigens for detecting specific antibodies]. / Ispol'zovanie leptospiroznykh mikrokapsul'nykh antigenov dlia vyiavleniia spetsificheskikh antitel.

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.

[A comparative evaluation of the sensitivity of different methods for the serological diagnosis of leptospirosis]. / Sravnitel'naia otsenka chuvstvitel'nosti razlichnykh metodov serologichicheskoi diagnostiki leptospiroza.

In this work the comparative evaluation of the sensitivity and serological specificity of the microcapsular agglutination (MCA) test, the passive hemagglutination (PHA) test and the microagglutination (MA) test are presented. In the MCA test leptospiral antigens, adsorbed on synthetic carrier capsules produced by Japan Lyophilization Laboratory, were used and the PHA test was made with the use of polyvalent erythrocyte diagnosticum. The study of blood serum samples from 46 leptospirosis patients revealed that the values of antibody titers in the PHA and MCA tests were 5.5-8.1 times higher than the traditional MA test. In the MCA and PHA tests antileptospiral antibodies could be detected as early as on days 1-3 of the disease when the results of the MA test were negative or very low. The maximum values of antibody titers in the MCA and PHA tests were detected on days 11-15 of the disease and in the MA test, on days 21-25. The MCA and PHA tests are genus-specific and permit the detection of antileptospiral antibodies irrespective of the serogroup of the infective agent. In the study of the blood sera of 40 patients with diseases of nonleptospiral etiology the MCA and MA tests yielded false positive results in 7.5% and the PHA test, in 12.5% of cases in titers below the diagnostic level. These data are indicative of high sensitivity and specificity of the serological tests used in this study.

Antibody activities of immunoglobulins in anti-leptospiral horse sera.

Antileptospiral sera from hyperimmunized horses were fractionated by gel filtration on Sephadex G-200 or by starch block electrophoresis. The fractions were examined quantitatively for leptospiricidal, agglutinating and complement fixing activities. The leptospiricidal activity was higher in the 78 globulin fraction than in the 19S globulin fraction, while the agglutinating activity was shared by both the fractions being higher in the 19S fraction. Complement fixing activity was found evenly in both the fractions. Leptospiricidal and complement fixing activities were higher in gamma-globulin than in T-globulin, while the agglutinating activity was comparable in the two globulin fractions. That is, the agglutinating activity may not always behave along with the leptospiricidal activity. The results were discussed in connection with the therapeutic antiserum product for human use.

Cross antigenicities of Leptospira interrogans serovar copenhageni Shibaura strain for preparing biological products in Japan.

Cross protective antigenicities of Leptospira were studied with 16 antisera to 13 serovars of Leptospira interrogans and a virulent strains "Shibaura (V)", which is now considered to belong to serovar copenhageni, for challenge. The antisera of the rabbits highly immunized with various serovar live antigens were examined for leptospiricidal activity; cross immunity was recognized only in the same serogroup, Icterohaemorrhagiae. Serovar icterohaemorrhagiae lacks a prt of the antigenic components of copenhageni, but the antiserum to the former showed potent leptospiricidal activity to Shibaura (V). Anti-copenhageni immune serum absorbed with the icterohaemorrhagiae antigen did not show any protective activity to Shibaura (V).

Skin reaction to lipids from avirulent strain Shibaura of Leptospira interrogans serovar copenhageni.

Sonically disrupted cells from avirulent strain Shibaura of Leptospira interrogans serovar copenhageni induced a skin reaction characterized by infiltration of polymorphonuclear leukocytes (PMN) associated with some edema in guinea pigs. To determine the substance inducing infiltration of PMN, lipids of avirulent strain Shibaura were extracted with chloroform--methanol--water after washing with acetone. The lipids comprised 28% of the dry weight of the cell. When the lipids were further separated into water--methanol and chloroform fractions, the most severe PMN infiltration of all samples was seen in the skin inoculated with extract recovered from the chloroform fraction. Neutral and polar lipids were detected after thin-layer chromatography of the chloroform extract. Neutral lipids were detected as free fatty acids (FFA). Fatty acids contained in polar lipids were mainly palmitic acid and palmitoleic acid, whereas FFA comprised 66.5% oleic acid. Skin reactions consisting of marked edema with mild infiltration of PMN were elicited by FFA. There was no obvious difference between a commercially available FFA mixture and the FFA from avirulent strain Shibaura. These observations suggest that FFA may play some role in the pathogenesis of leptospirosis.

Distribution of leptospirosis among stray dogs in the Okinawa Islands, Japan: comparison of the microcapsule and microscopic agglutination tests.

Three hundred and thirty-eight sera collected from stray dogs in the Okinawa islands were examined for antibodies against Leptospira interrogans using the microscopic agglutination test (MAT) and the one-point microcapsule agglutination test (MCAT). Seventy-eight sera (23%) showed a positive reaction to at least one of the six serovar antigens, and 69 of these reacted with serovar canicola by microcapsule agglutination test. The mixed microcapsule agglutination test detected 68 of the microscopic agglutination test-positive sera, and the 10 remaining were negative by microcapsule agglutination test. On the other hand, a single microcapsule agglutination test which was sensitized with serovar canicola detected 77 of the microscopic agglutination test-positive sera and the remaining one was microcapsule agglutination test-negative.

Studies on the potency test of antiserum for Weils disease by the intracutaneous method.

A method for estimating the leptospiricidal activity of therapeutic antiserum for Weil's disease was improved by using the intracutaneous method in guinea pigs. The neutralization curves between the leptospiral suspensions for challenge and the antisera were shown to be linear over a wide range of the dosis of the pathogen. Although the reproducibility of the neutralization experiments was high, a significant divergence was observed among the slopes in various test samples. However, the trouble due to such a discrepancy in the slope may practically be reduced if such an antiserum preparation that has a regression coefficient close to the mean of those of various neutralization lines constructed by many different products is adopted as the reference. A practical method for the potency test was suggested.

Comparison of antibody titers against borrelial strains isolated in Japan by the microcapsule agglutination test for serological studies of early Lyme disease.

Results of a passive microcapsule agglutination test using 2 Japanese strains, Borrelia garinii and Borrelia afzelii, were comparable to that of the passive microcapsule agglutination test using a Swiss strain Borrelia burgdorferi IRS in detecting antibodies of an early stage of Lyme disease patients in Japan. MCAT with microcapsule sensitized strain IRS seems likely to become one of the important tools for the early diagnosis of Lyme disease in the world. The highest titer of MCAT was detected in the serum samples of patients which were taken around 2 weeks after erythema chronicum migrants. The test is very simple and can be employed easily in diagnostic laboratories.

Development of a simple serological method for diagnosing leptospirosis: a microcapsule agglutination test.

A passive microcapsule agglutination test for the diagnosis of leptospirosis was developed by utilizing chemically stable microcapsules instead of sheep erythrocytes. In the test, sonically disrupted antigens of leptospira were sensitized to microcapsules treated with glutaraldehyde. Compared with the microscopic agglutination test, the passive microcapsule agglutination test showed a relatively genus-specific tendency and a 4- to 32-fold-higher sensitivity. The sensitized microcapsule antigens were stable for at least 1 year. The microcapsules coupled with mixed antigens can be used as a serodiagnostic screening test for diseases caused by various types of leptospira. The test, which is very simple and reproducible and requiring no specific training, can be employed easily as a routine test in diagnostic laboratories.

Intracutaneous infection with Leptospira icterohaemorrhagiae (Shibaura strain) of the guinea pig.

Experimental leptospirosis with Leptospira icterohaemorrhagiae Shibaura strain was studied in guinea pigs. When the pathogen was inoculated intracutaneously to the back of the animals, localized haemorrhage was observed at the inoculated site before the appearance of general haemorrhage. The severity of the local lesion increased progressively until the 7th day of inoculation. The minimum infective dose (MID) or the 50% infective dose (ID50) of the leptospiral suspension was determined by the appearance of the macroscopic local haemorrhage 7 days after inoculation. The MID thus determined was almost comparable with the value determined by the development of general symptoms and signs by conventional ip inoculation. The number of the pathogen per ID50 varied between 6 and 35 in five experiments. The local haemorrhage was effectively protected by active or passive immunization. Microscopically, haemorrhage at the inoculated site was found mainly in the dermis, directly beneath the epidermis in particular, and accompanied with leakage of the pathogen. The pathogen was also detected abunduntly in the thickened epidermal layer covering the inoculated area as well as in the epithelial matrix of hair-follicle, probably due to the proliferation of the pathogen at the site.

Detection of antibodies to leptospirosis in experimentally infected dogs using the microcapsule agglutination test.

Six puppies were infected with a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae and another five animals with a virulent strain of Leptospira interrogans serovar canicola, respectively. Antibodies were examined at 3, 5, 7, 11 and 14 days after infection, using the microcapsule agglutination test (MCAT) and the conventional microscopic agglutination test (MAT). Compared with the MAT, the MCAT detected early specific IgM antibody with high sensitivity. The MCAT titres reached a peak at the 7th day after infection and declined gradually after the 11th day, while the MAT titres increased up to the 14th day.

Seroprevalence of leptospirosis in a rural flood prone district of Bangladesh.

Leptospirosis is a worldwide zoonotic disease. In the present investigation, a total of 89 human sera from a flood prone district of Bangladesh was screened by a one-point microscapsule agglutination test (MCAT). MCAT-positive and -doubtful sera were further tested by microscopic agglutination test (MAT) against 16 reference serovars of Leptospira interrogans, and the antibody titres determined. In MCAT, 34 sera were positive and 22 were doubtful. Among those positive and doubtful sera, 33 and 20, respectively were tested by MAT. Thirty-four out of 53 MCAT-screened samples were MAT-positive. The titres ranged from 20 to 1600 with antibodies to serovars copenhageni, australis, cynopteri and icterohaemorrhagiae being the most prevalent. Eleven MCAT-positive samples failed to react with any strains used by MAT, suggesting the presence of new or untested serovars. Among the MAT-positive samples, the presence of antibody against two or more serovars was more common than that of a single serovar. The present study suggests that rural people in Bangladesh are at high risk to leptospiral infection.

Evaluation of a passive microcapsule agglutination test for the screening of human leptospirosis.

Microcapsules, absorbed with two mixed antigens--each composed of 3 sonicated leptospira serovars--were developed in the past as a microcapsule agglutination test (MCA-LS) for the screening of clinical leptospirosis. For this study, fifty serum samples, taken at an earlier and at a later stage of illness from 25 Italian in-patients with clinical symptoms of leptospirosis, were tested with both the MCA-LS one-dilution test and the microscopic agglutination (MA) test, the confirmatory test for leptospirosis, with 18 leptospira strains circulating in Italy. Compared with MA, MCA-LS showed a sensitivity of 91.7%, and a specificity of 92.3%, including leptospirosis not sustained by the diagnostic strains used in the MCA-LS kit.

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura.

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura were analyzed by thin-layer chromatography, gas-liquid chromatography, gas-mass spectrometry and infrared absorption spectrometry. The virulent cells possessed a characteristic lipid pattern consisting of free fatty acid (FFA) (41.8%), one major unidentified phospholipid (14.8%), phosphatidylethanolamine (PE) (12.9%), cholesteryl ester (CE) (9.3%), lysophosphatidylethanolamine (LPE) (4.9%) and diphosphatidyl-glycerol (DPG) (1.1%). Various fatty acids such as hexadecanoic (26.9%), hexadecenoic (15.4%), octadecenoic (26.5%) and octadecadienoic (27.4%) acids were detected in the FFA. The fatty acid composition of the major unidentified phospholipid distinctly differed from those of other lipids including PE, LPE, DPG and CE, and comprised mainly tetradecadienoic (53.6%), tetradecatrienoic (14.0%) and octadecanoic (13.8%) acids. This phospholipid with a large amount of polyunsaturated fatty acids with chain lengths of 14 carbon atoms was detected only in the lipids of the virulent cells.

Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.

Epidemiological studies on leptospirosis in Chiang Mai (Thailand).

A total of 270 serum samples collected in Chiang Mai province were examined for antibodies against leptospira using the microscopic agglutination test (MAT). Four of 40 serum specimens from patients who visited the hospital with the common cold, were positive with a titre of 20. Twelve (10.4%) of the 115 samples in the Doi Saket district showed a positive reaction. Only 2 of 115 sera of school children in Chiang Mai city had antibodies. Specific serovars detected were Leptospira hebdomadis (5), L. australis (3), L. icterohaemorrhagiae (2), L. bataviae (2), and one each of L. canicola, L. javanica and L. pyrogenes. One case of mixed infection with L. hebdomadis and L. javanica, and L. autumnalis and L. australis were observed.

Serodiagnosis of leptospirosis in China by the one-point MCA method.

The one-point MCA method is very simple to perform and useful as a screening test in diagnosing leptospirosis in routine clinical laboratories. The kit, sensitized with six serovars occurring in Japan, was also useful in detecting serum antibodies of patients with leptospirosis in China.

One-point method for serological diagnosis of leptospirosis: a microcapsule agglutination test.

This paper describes a simple and rapid microcapsule agglutination (MCA) test. The results obtained by this new test have been compared with those obtained by the microtitre MCA and the microscopic agglutination (MA) test. The procedures required for the new test are easier and can be performed more rapidly than those necessary for the microtitre MCA test. Furthermore, the new test is more sensitive than the MA test in the early stages of leptospirosis. This new test appears satisfactory as a screening test for the early diagnosis of leptospirosis.

Evaluation of the one-point microcapsule agglutination test for diagnosis of leptospirosis.

We have developed a one-point microcapsule agglutination test (MCAT) for the serodiagnosis of leptospirosis. The MCAT kit was evaluated for use in humans by six WHO Collaborating Centres for Reference and Research on Leptospirosis. The laboratories classified their serum samples on the basis of the microscopic agglutination test (MAT) and the following screening tests: enzyme-linked immunosorbent assay (ELISA), macroscopic (slide) agglutination test, or the complement fixation test. The MCAT may in some instances give a positive result earlier in the course of the disease than MAT or the ELISA IgM; on the other hand, it did not detect antibodies against some serovars, for example, those of the Sejroe or Australis serogroup in Slovakia. In contrast, however, the MCAT detected antibodies to serovar hardjo (the same serogroup as Sejroe) in patients from the United Kingdom and the Russian Federation.