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Detection by indirect immunofluorescence of Fc receptors in cells acutely infected with Herpes simplex virus.

In attempting to use the indirect fluorescent antibody test (IFA) to measure antibodies to herpes simplex virus (HSV), we found that all human sera gave a positive reaction with Chang liver cells infected with type 1 (HSV). All sera gave equivalent titers of 320-640 for acetone-fixed cells and about 40 for live cells (membrane fluorescence) in the presence of fluorescein-labeled antisera to human Ig; none of the sera reacted with uninfected cells. The fluorescence seen in fixed cells was primarily cytoplasmic; some cells showed a diffuse fluorescence, obscuring the demarcation between the nucleus and cytoplasm. Purified IgG from antibody-negative human sera and a purified Fc fragment of IgG were positive both for cytoplasmic and membrane fluorescence, whereas F(ab')2, IgM and IgA were unreactive. The reaction was also seen when an antiserum conjugate specific for the Fc fragment of IgG was used. The reactive IgG was present in freshly prepared plasma and serum; it could not be removed from serum either by ultracentrifugation or by serial absorption with HSV-infected cells. These findings suggest that the nonspecificity of the IFA results from the formation of low-avidity bonds between the large mass of native serum IgG and an Fc receptor on the plasma membrane and in the cytoplasm of cells infected with HSV. The results also suggest that extreme caution be exercised in attempting to use the IFA in the serodiagnosis of infections with HSV and perhaps the other human herpes-viruses.

Animals↗

Adverse neurologic reactions to the sting of the imported fire ant.

While the cutaneous manifestations of the imported fire ant (IFA) sting are well known, neurologic sequelae are much more unusual. We report 2 cases of grand mal seizures associated with the sting of the IFA. The first patient suffered numerous stings without evidence of systemic reactions prior to the onset of the seizure. The second patient had manifestations of anaphylaxis prior to the occurrence of seizure. In both cases, the onset of seizures was delayed. As the geographic range of the IFA increases and more patients are stung, unusual reactions will be seen in greater frequency.

Adolescent↗

Serological studies of peptostreptococci using an indirect fluorescent antibody test.

Members of the genus Peptostreptococcus are frequent isolates from periodontal lesions. A study was undertaken for determination of the serological relationships of oral and non-oral strains of several species of this genus. Eighty-nine strains of peptostreptococci representing seven species were tested by means of an indirect fluorescent antibody technique (IFA), with rabbit antisera to Peptostreptococcus anaerobius ATCC 27337, Peptostreptococcus micros VPI 2618-A, and Peptostreptococcus productus ATCC 27340. Each antiserum showed positive fluorescence when reacted with homologous cells. When anti-P. micros VPI 2618-A serum was added to suspensions of P. micros ATCC 33270 and clinical isolates of P. micros in the IFA, positive fluorescence was observed to a titer of 1:64 with 26 out of 29 strains. Positive fluorescence was also seen when P. anaerobius VPI 5737 and clinical isolates of P. anaerobius were tested with anti-P. anaerobius ATCC 27337 serum; a titer of at least 1:64 was observed in 10 out of 14 strains. These results support the presence of two serological groups of P. anaerobius, rabbit anti-P. anaerobius ATCC 27337 serum reacting with Group II organisms. No interspecies cross-reactivity was observed except with four strains of Peptostreptococcus magnus, which reacted with several antisera as well as with normal rabbit serum and the saline controls. These results indicate that rabbit antisera and the use of an IFA are useful in the identification of P. anaerobius and P. micros from oral lesions.

Fluorescent Antibody Technique↗

Enzyme-linked immunosorbent assay, immunofluorescent assay, and recombinant immunoblotting in the serodiagnosis of early Lyme borreliosis.

Serum samples from Bulgarian patients with physician-diagnosed erythema migrans (EM) (n=105) were examined using Borrelia burgdorferi ELISA (Boehring, Germany) after previous absorption with Treponema phagedenis. For IgM antibody detection sera were additionally pretreated with anti-IgG serum (RF absorbent). Serum samples of 93% of persons from healthy control group were IgM negative and all were IgG negative. Out of 105 patients with EM, 49% were IgM positive and 14 % were borderline. IgG ELISA showed positive results for 17% and borderline for 6% of the patients. Positive and borderline serum samples were examined further by immunofluorescent assay (IFA) and immunoblot test with recombinant B. burgdorferi proteins from strain PKo (B. afzelii) - p100, flagellin, OspA and OspC, and internal flagellin fragments from strains PKo and PBi (B. garinii) [B.Wilske, V.Fingerle, P. Herzer et al. 1993. Med. Microbiol. Immunol. 182:255]. IFA detected IgM antibodies against B. burgdorferi in 47 % of the positive and in none of the borderline by IgM ELISA serum samples as well as IgG antibodies in 83% of the positive and in 50% of the borderline by IgG ELISA samples. Presence of specific antibodies was confirmed by immunoblot in 71 % of the IgM ELISA postive and in 67 % of the IgG ELISA positive sera. In addition, anti-B. burgdorferi antibodies were detected in 60 % of the borderline by IgM ELISA serum samples. IgM serum reactivity was directed mainly against OspC antigen and flagellin and IgG antibodies were directed mainly against flagellin and p100. These findings clearly showed advantages of the ELISA test based on previous pretreatment of sera and capable to detect specific antibodies in more than half of patients with early Lyme borreliosis despite the well-known delayed immune response. IFA was less sensitive than ELISA in detection of anti-B. burgdorferi antibodies. An additional examination of ELISA borderline sera by immunoblot revealed more positive results. Serum reactivity to a single OspC antigen seems to be a sufficient criterion for positive IgM immunoblot.

Antibodies, Bacterial↗

Comparison of enzyme-linked immunosorbent assay, indirect fluorescent antibody test, and direct agglutination test for detecting Toxoplasma gondii antibodies in naturally aborted ovine fetuses.

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to greater than 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.

Abortion, Veterinary↗

Diagnosis of bovine viral diarrhea virus infection using monoclonal antibodies.

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.

Animals↗

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Development and evaluation of a recombinant antigen, monoclonal antibody-based competitive ELISA for heartwater serodiagnosis.

Cowdria ruminantium is the etiologic agent of heartwater, a tick-transmitted foreign animal disease with considerable potential for entrance into the USA. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect serologic responses to C. ruminantium infection. The cELISA utilized a recombinant form of the C. ruminantium major antigenic protein (MAP-1) as the antigen and an anti-MAP-1 monoclonal antibody as the competing indicator reagent. Experimental antisera to C. ruminantium and a wide variety of related ehrlichial organisms were used to evaluate cELISA reactivity. Only sera against C. ruminantium, Ehrlichia canis, E. chaffeensis, and a recently discovered cervine ehrlichia-like organism reacted positively in the cELISA. Specificity of the cELISA was > or = 99.5% in a survey of 1,774 southeastern US and Puerto Rican slaughter cattle sera but was only 85% in a group of 79 hunter-killed white-tailed deer (Odocoileus virginianus) from the southeastern USA. Reference true-positive and cELISA false-positive sera were further analyzed by end point titrations using the cELISA and by indirect fluorescent antibody (IFA) tests for reactivity with C. ruminantium, E. canis, and E. chaffeensis antigens. True heartwater-positive sera were significantly more reactive using the cELISA and C. ruminantium IFA procedures (P < 0.05), whereas false-positive sera were significantly more reactive with the antigens used in the E. chaffeensis IFA procedure (P < 0.05). A group of sera from 210 field-origin ruminants residing on known or potentially heartwater-endemic Caribbean islands revealed a substantial (12.4%) prevalence of cELISA-positive specimens. The cELISA is a relatively specific serodiagnostic test for heartwater in cattle and could be used to monitor for possible introduction of the disease into the USA. The cELISA may also be an excellent tool for monitoring the success of an ongoing Caribbean Amblyomma tick eradication program designed to eliminate the biological vector responsible for the perpetuation and spread of this dangerous foreign animal disease.

Animals↗

Production of recombinant protein Pap31 and its application for the diagnosis of Bartonella bacilliformis infection.

Tropical bartonellosis is a highly fatal epidemic and endemic infectious disease that occurs throughout the communities of the Andes Mountains in South America. The disease is caused by the facultative intracellular bacteria, Bartonella bacilliformis. The emergence of bartonellosis in new geographic areas and an increase in the number of reported cases suggest the need for a rapid test for epidemiologic study and investigation of the disease burden. The objective of this research is to develop a rapid serologic diagnostic test using recombinant antigens to overcome the limitations of the current standard IFA technique for laboratory diagnosis. Western blot analysis with patient sera of whole cell lysate separated on a 2D gel identified Pap31 as a dominant antigen. PCR primers were designed according to the sequence of ATCC strain 35685 to amplify the gene coding for Pap31 from a local isolate (HOSP 800-09, Peru). The amplicon was subsequently cloned into pET24a, adding the T7 tag, and expressed in E. coli. Patient sera with different IFA titers confirmed the diagnostic band of 31 kDa on a Western blot of SDS-PAGE. The performance of affinity-purified recombinant Pap31 (rPap31) was also evaluated in an ELISA format with 137 patient sera of known IFA titers. The range of ELISA reading from positive sera did not overlap with the range of those from negative sera, suggesting the potential application of rPap31 in both ELISA for high throughput regional hospital settings and in the construction of handheld rapid tests for rural clinical sites.

Antigens, Bacterial↗

Novel features of intermediate filament dynamics revealed by green fluorescent protein chimeras.

In order to study the dynamic behavior of intermediate filament networks in living cells, we have prepared constructs fusing green fluorescent protein to intermediate filament proteins. Vimentin fused to green fluorescent protein labeled the endogenous intermediate filament network. We generated stable SW13 and NIH3T3 cell lines that express an enhanced green fluorescent protein fused to the N-terminus of full-length vimentin. We were able to observe the dynamic behavior of the intermediate filament network in these cells for periods as long as 4 hours (images acquired every 2 minutes). In both cell lines, the vimentin network constantly moves in a wavy manner. In the NIH3T3 cells, we observed extension of individual vimentin filaments at the edge of the cell. This movement is dependent on microtubules, since the addition of nocodazole stopped the extension of the intermediate filaments. Injection of anti-IFA causes the redistribution or 'collapse' of intermediate filaments. We injected anti-IFA antibodies into NIH3T3 cells stably expressing green fluorescent protein fused to vimentin and found that individual intermediate filaments move slowly towards the perinuclear area without obvious disassembly. These results demonstrate that individual intermediate filaments are translocated during the collapse, rather than undergoing disassembly-induced redistribution. Injections of tubulin antibodies disrupt the interactions between intermediate filaments and stable microtubules and cause the collapse of the vimentin network showing that these interactions play an important role in keeping the intermediate filament network extended. The nocodazole inhibition of intermediate filament extension and the anti-IFA microinjection experiments are consistent with a model in which intermediate filaments exhibit an extended distribution when tethered to microtubules, but are translocated to the perinuclear area when these connections are severed.

3T3 Cells↗

Cutoff and target values for intra-abdominal fat area for prevention of metabolic disorders in pre- and post-menopausal obese women before and after weight reduction.

BACKGROUND: The Japan Society for the Study of Obesity originally proposed a cutoff value of >100 cm(2) for the intra-abdominal fat area (IFA) as a definition for "visceral fat obesity" in Japanese adults. There are no studies on the cutoff or target values after weight reduction in pre- and post-menopausal women. METHODS AND RESULTS: In the present study 149 pre-menopausal obese women (PreM, 43.3 years, 27.3 kg/m(2)) and 58 post-menopausal women (PostM, 53.9 years, 27.7 kg/m(2)) participated in a 14-week weight reduction program. The IFA was measured by computed tomography. The program induced significant reductions in body weight (8.6 kg in PreM and 7.8 kg in PostM). The IFA decreased significantly from 80.4 +/- 41.3 to 50.7 +/- 23.8 (PreM) and from 115.4 +/- 38.0 to 75.7 +/- 30.5 (PostM). CONCLUSIONS: The receiver-operating characteristic curve analyses revealed that the appropriate cutoff values were 80 cm(2) (PreM) and 110 cm(2) (PostM) before the program, and after the program the appropriate target values were determined as 60 and 70 cm(2), respectively.

Abdomen↗

Transmission experiments of cilia-associated respiratory bacillus in mice, rabbits and guineapigs.

Transmission experiments of cilia-associated respiratory (CAR) bacillus were performed in mice in order to clarify the principal route of the infection, and in rabbits and guineapigs in order to examine their susceptibility. Determination of the infection was evaluated serologically by the indirect immunofluorescence assay (IFA) technique and histologically by the presence of CAR bacillus in the airways. BALB/c mice were intranasally inoculated with the SMR strain of CAR bacillus. The IFA antibody to the bacteria in these mice rose to more than 1:160 at 4 weeks postinoculation (PI) and the mice were utilized as transmitters for the following experiments. One out of 15 uninfected mice kept in intracage contact with infected mice became infected from 4 weeks after contact. Incidence of contact infection increased thereafter. On the other hand, there was no evidence of infection in the uninfected mice housed in the separate cages from the cage in which infected mice were housed throughout the 12-week observation period. The primary method of CAR bacillus transmission seems to be direct contact with infected mice or fomites contaminated by infected mice; airborne transmission appears to be of little importance. Rabbits and guineapigs were also intranasally inoculated with the SMR strain of CAR bacillus. IFA antibodies were positively detected by 4 weeks PI, but no CAR bacillus nor histological changes relating to the infection were observed in the airways of either species. It is suggested that rat origin CAR bacillus can transmit to rabbits and guineapigs, and that the infection can spread to other species of rodents and rabbits.

Animals↗

Application of enzyme-linked immunosorbent assay for the seroepizootiological survey of antibodies against porcine cytomegalovirus.

Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against porcine cytomegalovirus (PCMV) was developed using the OF-1 strain of PCMV as an antigen. Results of the ELISA were compared to those of indirect fluorescent antibody (IFA) and serum neutralization (SN) tests. ELISA and IFA test were found to be extremely sensitive more than SN test. All of 11 tested sera were highly reactive in both ELISA and IFA test, but 6 of them were antibody-negative in the SN test. The retrospective survey of 436 fattening pig sera collected in Japan in 1981 showed that 433 (99.4%) of them were highly antibody-positive in ELISA.

Animals↗

Demonstration of common antigens on cell surface of Clostridium chauvoei and C. septicum by indirect-immunofluorescence assay.

The common antigens between Clostridium chauvoei and C. septicum were examined by indirect-immunofluorescence assay (IFA). Antisera to formalized cells of C. chauvoei and C. septicum strains and to EDTA-soluble antigens of these strains were used. The antisera to formalized cells, which have reacted only with homologous antigens in agglutination tests, reacted not only with homologous antigens but also with heterologous antigens in IFA. The antisera to EDTA-soluble antigens, which have shown no reactivities in somatic agglutination tests, reached with both homologous and heterologous antigens in IFA. These results indicate that these species possess common antigens, which are undetected by agglutination tests, on the cell surface and a some of them are solubilized by EDTA-treatment.

Antigens, Bacterial↗

Western blotting using recombinant Hantaan virus nucleocapsid protein expressed in silkworm as a serological confirmation of hantavirus infection in human sera.

Recombinant Hantaan virus nucleocapsid protein expressed in silkworm larvae was applied as a serological diagnostic antigen in Western blots (WB) of human sera. The sensitivity of this method was similar to that of the IFA test. Hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica diagnosed by their cross-reactivity in WB. The specificity of this method was higher than that of IFA test because the background was low. Sera that exhibited high background staining in the IFA test were readily diagnosed with this method. We recommended WB using recombinant Hantaan virus nucleocapsid antigen as a confirmatory procedure for the serodiagnosis of hantavirus.

Animals↗

Detection of an autoantibody from Pug dogs with necrotizing encephalitis (Pug dog encephalitis).

An autoantibody against canine brain tissue was detected in the cerebrospinal fluid (CSF) and serum of two Pug dogs (Nos. 1 and 2) by indirect immunofluorescence assay (IFA). Dog No. 1, a 2-year-old male, exhibited severe depression, ataxia, and generalized seizures and died 2 months after the onset of symptoms. Dog No. 2, a 9-month-old male, exhibited severe generalized seizures and died 17 months after the onset of symptoms. Histopathologic examination revealed a moderate to severe multifocal accumulation of lymphocytes, plasma cells, and a few neutrophils in both the gray and white matter of the cerebrum in dog No. 1. In dog No. 2, the cellular infiltrates were mild, but there was a severe, diffuse, and multifocal necrosis in the cerebral cortex with prominent astrocytosis. With the aid of IFA using fluorescein isothiocyanate-labeled antidog IgG goat serum and a confocal imaging system, specific reactions for glial cells were detected in the CSF of these Pug dogs but not in six canine control CSF samples. Double-labeling IFA using CSF from these Pug dogs and a rabbit antiserum against glial fibrillary acidic protein (GFAP) revealed that the autoantibody recognized GFAP-positive astrocytes and their cytoplasmic projections. By immunoblot analysis, the autoantibody from CSF of these Pug dogs recognized two common positive bands at 58 and 54 kd, which corresponded to the molecular mass of human GFAP. The role of this autoantibody for astrocytes is not yet clear. However, if the presence of the autoantibody is a specific feature of Pug dog encephalitis, it will be a useful clinical diagnostic marker and a key to the pathogenesis of this unique canine neurologic disease.

Animals↗

Analyzing the H19- and T65-epitopes in 38 kd phosphorylated protein of Marek's disease viruses and comparing chicken immunological reactions to viruses point-mutated in the epitopes.

DNA sequencing analysis in 38 kd phosphorylated protein (pp38) ORF of Marek's disease viruses (MDV) indicated that all tested 10 virulent strains with different pathotypes had 'A' at base #320 and glutamine at aa#107 while reacted with monoclonal antibody (Mab) H19 in indirect fluorescence antibody test (IFA). However, vaccine strain CVI988 had 'G' at base#320 and arginine at aa#107 instead, when it was negative in IFA with Mab H19. Some strains were also reactive with Mab T65 in IFA while there was 'G' at base #326 and glycine at aa#109, but the other strains, which had 'A' at base #326 and glutamic acid at aa#109, did not react with Mab T65. By comparison of CVI988 to its point mutants CVI/rpp38(AG) and CVI/rpp38(AA) with 1 or 2 base(s) changes at bases #320 and /or #326 of pp38 gene for their reactivity with Mab H19 and T65, it was confirmed that the glutamine at aa#107 and glycine at aa#109 were critical to epitopes H19 and T65 respectively. Immuno-reactions to MDV were compared in SPF chickens inoculated with cloned CVI988 and its mutant CVI/rpp38(AG). It was found that antibody responses to MDV in chickens inoculated with CVI/rpp38(AG) were delayed and significantly lower than that in chickens inoculated with the native CVI988. By differential comparison of antibody titers to different antigens, a third epitope specific to CVI988 and dependent on arginine at aa#107 was suggested to be responsible for the big difference in antibody responses induced by native CVI988 and its mutant.

Animals↗

Establishment of an enzyme-linked immunosorbent assay for detection of hantavirus antibody of rats using a recombinant of nucleocapsid protein expressed in Escherichia coli.

A recombinant nucleocapsid protein of Hantaan virus (HTN) 76-118 strain expressed in E. coli was applied as a serodiagnostic antigen in an enzyme-linked immunosorbent assay (rHTN-ELISA) for detection of hantavirus antibody in rat sera. The sensitivity and specificity of the rHTN-ELISA were compared with those of the indirect immunofluoresent assay (IFA) using virus-infected cells. The sensitivity of rHTN-ELISA was similar to that of the IFA both in experimentally SR-11 infected rat and naturally infected rat sera. Sera showing a low antibody titer in IFA and suspected to be negative by other methods were also found to be negative in rHTN-ELISA. These results indicate that rHTN-ELISA is effective as a screening method for serodiagnosis of hantaviruses, because of its high sensitivity, specificity, safety and suitability for processing large number of samples.

Animals↗