Evaluation of an indirect hemagglutination inhibition technique for identifying antibody in animals experimentally and naturally exposed to fungi

Rabbits were exposed to aerosols containing spores of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, or of a Penicillium sp. Sera from these rabbits were tested by indirect hemagglutination (IHA) and by IHA inhibition. The serologic reactions with the rabbit sera were compared to reactions with sera from cattle naturally exposed to airborne microorganisms. By three months of age, most cattle had positive IHA reactions to A. fumigatus and Penicillium antigens. The IHA inhibition tests indicated that antibody production in 12 of the 20 cattle probably resulted from exposure to A. flavus. One calf reacted as if sensitized by A. niger. Two were totally nonreactive. Five of the cattle had reactions that were not identifiable relative to the reactions in rabbits.Purchased by U.S. Department of Agriculture for Official Use.     

Indirect haemagglutination (IHA) test in the serodiagnosis of amoebiasis

Among 72 patients clinically suspected of Entamoeba histolytica (E. histolytica) infections, 39 positive cases (54%) were detected serologically by the indirect hemagglutination (IHA) test. Parasitologically, microscopic examination of three consecutive stool specimens from all these patients indicated positivity for E. histolytica cysts and or trophozoites in 10 of the patients with IHA antibody titers greater than or equal to 1:128, which is of clinical significance. Another 2 patients were parasitologically positive but showed low IHA antibody titres (1:32-1:64); follow up indicated response to treatment with metronidazole. The highest serological positivity (100%) were detected in patients with liver abscess, all were clinically proven cases of extra-intestinal amoebiasis. IHA antibody levels of clinical significance were seen in all four patients with chronic dysentery with parasitological evidence of E. histolytica in their stools. In a group of patients with abdominal pain nine positives were detected serologically, four of which were positively diagnosed concurrently by parasitology; the remaining five patient's sera showed high IHA antibody titres with absence of cysts or trophozoites in stools, indicative possibly of persistence of antibodies from past infection. The serologic determination of E. histolytica IHA antibodies in a control group consisting of normal healthy school children and adults of both sexes without any clinical evidence of amoebiasis showed the absence of any positive titres of clinical significance; low titres (1:32-1:64) were detected in 5.2% of 232 sera tested. Parasitological examination of three consecutive stool specimens from all individuals in the control group showed the presence of cysts of E. histolytica in just two among 232 tested (0.9%).     

Magnetic microbead-based enzyme-linked immunoassay for detection of Schistosoma japonicum antibody in human serum

A specific and sensitive immunoassay based on magnetic microbead separation for schistosomiasis japonica screening is presented in this article. So far as we know, this is the first time that magnetic microbead-based enzyme-linked immunoassay (MEIA) has been used for the determination of Schistosoma japonicum (Sj) antibody in human serum. Fluorescein isothiocyanate (FITC)-labeled soluble egg antigen (SEA) and polymer-coated magnetic beads, to which anti-FITC monoclonal antibodies were immobilized, were used as separation support in MEIA. Immunoassay parameters were optimized based on a direct immunoreaction of SEA on the magnetic microbead and Sj antibody in serum samples. The laboratory experimental results showed that the MEIA method was more sensitive and more precise than traditional SEA-ELISA (enzyme-linked immunosorbent assay). In the field test, human sera collected from 513 infected humans and 2260 uninfected humans were tested with indirect hemagglutination assay (IHA), dipstick dye immunoassay (DDIA), and MEIA. IHA and DDIA were then compared with MEIA, and a lower false negative rate (0.97%) was obtained.     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

Evidence for a fifth (G) region in theH-2 complex of the mouse

Antisera (B10.129×A)F₁ anti-P and (B10×A)F₁ anti-B10.P contain antibodies that define, in the PVP hemagglutination test, an antigen originally described as G or H-2.7. Of the independentH-2 haplotypes, the H-2.7 antigen is present inf, j, k, p, ands. In addition, the antisera also contain a weak cytotoxic antibody, distinct from anti-H-2.7. The cytotoxic antibody reacts with antigens controlled by theK orI regions. The hemagglutinating H-2.7 antibody does not have cytotoxic activity. The genetic determinant coding for antigen H-2.7 can be mapped into the chromosomal segment between theS andD regions. The H-2.7 antigen thus serves as a marker for a new region of theH-2 complex. The locus coding for antigen H-2.7 is designatedH-2 G and the correspondingH-2 regionG. The H-2.7 antigen has a tissue distribution distinct from that of the H-2 antigens controlled by theK orD regions. So far it could be detected primarily on erythrocytes.     

Serologic test for antibody to Sarcocystis in cattle.

Soluble antigen was prepared from Sarcocystis zoites obtained from heart muscle of a bovine inoculated with sporocysts from canine feces and killed 120 days after infection. The antigen was used in an indirect hemagglutination (IHA) test and an agar gel diffusion test to detect antibody to Sarcocystis in experimentally infected cattle. IHA serum titers began to rise 30 to 45 days after infection and reached levels up to 1:39,000 90 days after infection. Sera collected under field conditions from 21 dairy cows had IHA titers ranging from 1:54 to 1:486. Since all cows appeared in good health, titers of 1:486 or less can probably be considered nonsignificant with regard to diagnosis of clinical disease. No positive Sarcocystis IHA titers were obtained with human sera previously found to be IHA positive for toxoplasma, indicating a lack of cross reactivity between antigens. Precipitins in the agar gel diffusion test appeared 30 days postinoculation and became very pronounced at 65 to 90 days.     

Induction of humoral antibody response by soluble polysaccharide of Cryptococcus neoformans

Mice were immunized with purifiedCryptococcus neoformans soluble polysaccharide, or with the polysaccharide coupled to methylated bovine serum albumin or methylated bovine gamma globulin. The polysaccharide-methylated protein complexes were no more immunogenic than the purified polysaccharide when used without Freund's incomplete adjuvant; however, the methylated protein complexes induced higher levels of antibody than purified polysaccharide when emulsified in the adjuvant. Sera from mice were titrated by passive hemagglutination, and maximum antibody titers were observed 14 to 21 days after immunization. Antibody titers declined rapidly after 14 to 21 days in mice immunized with antigen alone; whereas, animals immunized with cryptococcal antigen emulsified in adjuvant remained at peak levels throughout a six week experimental period. All antigens were immunogenic over a wider dosage range when contained in adjuvant. Individual mice immunized with an adjuvant emulsion of purified polysaccharide varied widely in the amount of antibody produced, with some of the animals producing no detectable antibody.     

An indirect hemagglutination antibody test to detect antibodies to Borrelia burgdorferi in patients with Lyme disease

An indirect hemagglutination antibody (IHA) test was evaluated for its ability to detect borrelial antibodies in serum samples from patients with Lyme disease. The key test reagent developed for this antibody detection system was tannic acid-treated and glutaraldehyde-fixed sheep red blood cells (SRBC) containing Borrelia burgdorferi (Bb) antigens attached to the outer surface of the SRBC. In order to establish suitable cut-off titers, initial specificity and sensitivity measurements were made using sera from 100 anonymous healthy volunteers and 30 additional pre-determined samples known to be non-reactive or reactive for Lyme disease or syphilis. These results were compared with those obtained using a commercially available ELISA. At titers >/=64, the IHA test had a combined 98% specificity and 100% sensitivity for these 130 serum samples, 30 of which were known positives or negatives, whereas the ELISA was less specific (93%) and much less sensitive (80%). Subsequent testing was performed on sera from 65 patients with the erythema migrans (EM) rash and 20 patients with early disseminated (cardiac/neurologic) symptoms or with Lyme arthritis. At initial presentation, 46-48% of the EM patients had IHA reactivity, with titers >/=128, while 42% were positive in the ELISA. Follow-up testing performed on these EM patients, 8-12 days after receiving antibiotic treatment, revealed that Bb antibodies were detected best by the IHA test (83-86% reactive) relative to the ELISA (81% reactive). Bb antibodies were readily detectable on all of the serum samples from the early disseminated and late stage Lyme disease cases in both assay systems. Based on these results and because of its technical and interpretive simplicity, the IHA test should be considered as a useful and convenient alternative for the serological analysis of Bb infections.     

Serosurveillance for Pseudomonas pseudomallei infection in Thailand.

A nation-wide survey was conducted to see the prevalence of serosensitivity to Pseudomonas pseudomallei antigens by indirect hemagglutination (IHA) and indirect immunofluorescent assay (IFA) for IgG and IgM. Serum samples were collected from blood donors in eight selected areas and bacteriologically confirmed melioidosis patients in Ubon Ratchathani province. The distribution patterns of antibody titers were compared among the survey areas with cut-off points set at 1:160 for IHA, 1:4 for IFA-IgM and 1:32 for IFA-IgG. These cut-off points were decided by ROC (Receiver Operating Characteristics) analysis. The specificity (% true negative reactions) of each serological test in the general population differed significantly among survey areas, possibly reflecting the extent of inapparent infection in each community. IFA was more successful than IHA in differentiating between negative from positive reactions. The survey classified the areas into endemic (Khon Kaen, Ubon Ratchathani), transported (Bangkok), and non-endemic (other provinces) types.     

鼠疫菌F1抗体/抗原酶联免疫诊断试剂盒的应用评价

目的对兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒进行临床应用评价。方法采用双抗原/抗体夹心酶联免疫吸附试验(ELISA)、间接血球凝集试验(IHA)、胶体金免疫层析试验(GICA)3种方法的诊断试剂对比检测云南省地方病防治所中心实验室保藏的和现场采集的血清样品和脏器样品,对血清样品做鼠疫菌F1抗体检测,对脏器样品做鼠疫菌F1抗原检测。结果在358份血清样品中,ELISA试剂检出F1抗体阳性52份(14.52%),IHA试剂检出阳性37份(10.34%),GICA试剂检出阳性45份(12.57%)。ELISA与IHA试剂的符合率为95.23%,与GICA试剂的符合率为96.92%。经统计学χ2检验,ELISA试剂检出F1抗体阳性率高于IHA试剂(χ2=11.53,P=0.000 7),与GICA试剂检出的差异无统计学意义(χ2=3.27,P=0.070 4)。进一步分析滴度差值频数,ELISA试剂检测人血清的敏感性高于IHA试剂的样品占87.5%。在117份脏器样品中,3种试剂均检出F1抗原阳性15份(12.82%),符合率100%。滴度差值频数比较,ELISA试剂检测敏感性高于反向间接血球凝集试验(RIHA)试剂的样品为78.57%。结论兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒性质特异,其敏感性优于IHA试剂盒和GICA试剂条,值得在鼠疫的监测和快速诊断中推广应用。     

An enzyme-linked immunosorbent assay to detect alkali-soluble spore surface antigens of strains of Nosema bombycis (Microspora: Nosematidae)

Spore surface antigens of strains of Nosema bombycis were extracted with alkaline solutions and used in an indirect enzyme-linked immunosorbent assay. Treatment of N. bombycis spores with 0.1 n potassium carbonate or potassium hydroxide solution at 27°C for 30 min was sufficient for the extraction of the antigens. Usually, 10⁸ spores of N. bombycis liberated ca. 30 μg spore surface proteins. The indirect enzyme-linked immunosorbent assay detected as little as 60 ng of spore surface proteins (ca. 2000 spore-equivalent antigen). The alkali-soluble spore surface antigens of N. bombycis contained a specific antigen and were stable under storage at −20°C for more than 1 year. The serological assay separated the Nosema isolates pathogenic to the silkworm into three groups.     

Trypanosoma musculi: Passive hemagglutination technique to measure antibody in mice

A passive hemagglutination assay was developed to measure Trypanosoma musculi-specific antibody in mice. Indicator-erythrocyte donor mice received 550 rad ⁶⁰Co 24 hr before intraperitoneal injection of 3 × 10⁴T. musculi. T. musculi antigen-coated erythrocytes were obtained from these mice on Day 9 postinfection. T. musculi antigen-coated erythrocytes obtained in this manner were used as indicator erythrocytes in a passive hemagglutination procedure. Serum from hyperimmunized mice (three consecutive infections at 21-day intervals) gave titers as high as 1:1024. Titers of 1:256 and 1:512 were obtained from singly infected mice on Days 18 and 28 postinfection, respectively. In marked contrast, nude mice infected with T. musculi did not produce a detectable agglutinating antibody response. Erythrocytes obtained from either irradiated (550 rad ⁶⁰Co) uninfected mice, nonirradiated infected mice, or normal mice did not agglutinate when combined with any of the sera tested. These data suggest the usefulness of this passive hemagglutination assay for the measurement of antibody to T. musculi in the serum of infected mice.     

Experiments on inter-specific competition between three species of Tachinids attacking the sugar cane moth borer,Diatraea saccharalis (F.)

Three species of Tachinids (Lixophaga diatraeae T.T.,Paratheresia claripalpis van der Wulp, andMetagonistylum minense T.T.) were used to study intrinsic larval competition under laboratory conditions in Barbados. When equal numbers of freshly hatched larvae ofLixophaga andParatheresia were simultaneously inoculated into fullgrown moth borer caterpillars, both parasite species survived in equal proportions. In simultaneous inoculations ofMetagonistylum andLixophaga the first-mentioned species was clearly inferior (ratio about 1∶2). In inoculations withMetagonistylum andParatheresia the intrinsic inferiority of the first-mentioned species was still more pronounced (ratio 1∶5). When maggots of all three species were inoculated simultaneously the ratio of parasite survival forLixophaga: Paratheresia: Metagonistylum was approximately 2∶2∶1, indicating again thatMetagonistylum is the inferior species, whereasLixophaga andParatheresia are equally competitive. The results described above did not change appreciably when one species was given a numerical advantage over the other, e.g. when three maggots ofMetagonistylum were simultaneously inoculated with only one maggot ofLixophaga (orParatheresia). In most of these tests,Lixophaga andParatheresia still remained the superior species. When maggots of one species were inoculated two days in advance of the other species — i.e. when one species had a developmental advantage — the earlier inoculated species usually survived in greater proportions than it would have in simultaneous inoculations. Under Barbados conditions the larval period ofLixophaga is the shorter (5 days), followed byMetagonistylum (6–7 days), and the longest larval period occurs inPara heresia (7–9 days). When a species with a faster development was inoculated two days in advance of a species with a slower development (e.g.Lixophaga two days in advance ofParatheresia), the developmental difference between the two species was magnified and the earlier inoculated species was then clearly the superior one. When, on the other hand, a species with slower development was given a two-day advantage in time the slower growing species survived in greater proportions than it would have done in simultaneous inoculations. The developmental advantage of its competitor was, in this case, eliminated.     

Serological diagnosis of petriellidiosis (Allescheriosis)

Soluble antigens in culture filtrates of three strains of Petriellidium boydii and three strains of Monosporium apiospermum were examined. Antigens were separated from concentrated crude filtrates by anion-exchange chromatography. A single major peak (Antigen 1), constituting a significant proportion of the total recoverable carbohydrate, was the only product isolated from each of four chromatographed filtrates. Depending on the fungus strain, Antigen 1 consisted of 90–96% carbohydrate, 3–4% protein, and 2–4% nucleic acid. Antigen 1 was found to consist of a population of molecules with a heterogeneous molecular size when assayed by gel filtration chromatography; however, isolated fractions of Antigen 1 proved to be immunologically identical when examined by Ouchterlony immunodiffusion. In addition, Antigen 1 from each strain was immunologically identical to similar preparations of Antigen 1 from the other five fungus strains. Chromatography of culture filtrates from two strains of M. apiospermum revealed a second peak (Antigen 2), which was found to consist of 70% carbohydrate, 16% protein, and 4% nucleic acid. Although Antigen 2 contained four times as much protein as Antigen 1, the two preparations were immunologically identical by immunodiffusion tests. Ion-exchange chromatography proved to be a useful procedure for isolating antigens of P. boydii and M. apiospermum from culture filtrates.     

Septoglomus jasnowskae and Septoglomus turnauae,two new species of arbuscular mycorrhizal fungi (Glomeromycota)

Phylogenetic analyses of SSU-ITS-LSU nrDNA sequences and morphological studies of spores and mycorrhizae confirmed our supposition of finding two new species of arbuscular mycorrhizal fungi of the genus Septoglomus in the phylum Glomeromycota. Morphologically, the first species, named S. jasnowskae, is distinguished by its pale yellow to brownish yellow, small spores with a 2-layered spore wall, of which the colourless outer layer 1 stains dark in Melzer’s reagent and layer 2 is laminate. The spores usually arise in loose clusters. The structures most distinguishing S. turnauae are its two coloured laminate layers in the 4-layered spore wall. In the field S. jasnowskae was associated with roots of Ammophila arenaria and an unrecognized plant species colonizing maritime dunes of the Mediterranean Sea near Thessalonica (Greece) and Calella (Spain), respectively, and S. turnauae formed mycorrhiza with a Cistus sp. (Cistaceae) growing in the soil of a mine located in Sulcis-Iglesiente, SW-Sardinia, Italy. In single-species cultures with Plantago lanceolata as host plant, the mycorrhiza of S. jasnowskae consisted of arbuscules, hyphae and vesicles, and that of S. turnauae comprised arbuscules and hyphae only.     

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

Production of high titre antibody response against Russell's viper venom in mice immunized with ethanolic extract of fruits of Piper longum L. (Piperaceae) and piperine

Piper longum L. fruits have been traditionally used against snakebites in north-eastern and southern region of India. The aim of the study was to assess the production of antibody response against Russell's viper venom in mice after prophylactic immunization with ethanolic extract of fruits of Piper longum L. and piperine. The mice sera were tested for the presence of antibodies against Russell's viper venom by in vitro lethality neutralization assay and in vivo lethality neutralization assay. Polyvalent anti-snake venom serum (antivenom) manufactured by Haffkine Bio-Pharmaceutical Corporation Ltd. was used as standard. Further confirmation of presence of antibodies against the venom in sera of mice immunized with PLE and piperine was done using indirect enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion test. Treatment with PLE-treated mice serum and piperine-treated mice serum was found to inhibit the lethal action of venom both in the in vitro lethality neutralization assay and in vivo lethality neutralization assay. ELISA testing indicated that there were significantly high (p < 0.01) levels of cross reactions between the PLE and piperine treated mice serum and the venom antigens. In double immunodiffusion test, a white band was observed between the two wells of antigen and antibodies for both the PLE-treated and piperine-treated mice serum. Thus it can be concluded that immunization with ethanolic extract of fruits of Piper longum and piperine produced a high titre antibody response against Russell's viper venom in mice. The antibodies against PLE and piperine could be useful in antivenom therapy of Russell's viper bites. PLE and piperine may also have a potential interest in view of the development of antivenom formulations used as antidote against snake bites.     

Panchanania andPhragmospathula,two new genera of the Hyphomycetes

Two new Hyphomycetes collected from Jaipur, Rajasthan, India are described.Panchanania jaipurensis gen. et sp.n., collected on dead and dying twigs ofGrewia salvifolia has characteristic solitary stalked blastospores, each with a dark-coloured head and paler stalk, on sympodulae borne on synnemata.Phragmospathula phoenicis gen. et sp.n., collected on dead leaf rachis ofPhoenix sp. has characteristic solitary spathulate phragmospores (blastospores) which are dark-coloured in the middle and paler at the ends and are produced on short conidiophores capable of proliferation from within. On the basis of the mode of development of the spores, both fungi are placed in the Torulaceae Corda emend. Subram.     

In Vivo Production of Neuraminidase by Pasteurella haemolytica in Market Stressed Cattle After Natural Infection

Pasteurella haemolytica (Ph) is the most important cause of the bovine acute fibrinohemorrhagic pneumonia that occurs in market stressed calves after shipment to feedyards. Recent characterization of neuraminidase production by these organisms has shown that all 16 serotypes produce an immunologically similar form of the enzyme. Anti-neuraminidase antibody against PhA1 and PhA6 was determined in 101 2- to 5-month-old calves, on their farms of origin, at the order buyer barn (OBB), and through 28 days in the feedyard. Half of the calves were vaccinated with a killed Ph serotype-A1 (PhA1) product. Nasal secretion and tonsil wash specimens were cultured for Ph and Pasteurella multocida (Pm). Serum antibody against PhA1 and PhA6 was measured by indirect hemagglutination (IHA), and anti-neuraminidase antibody was determined by the neutralization assay. At the feedyard, 73 calves had respiratory tract disease. IHA values ranged between 1:2 and 1:1024 for PhA1 and between 1:2 and 1:512 for Ph serotype A6 (PhA6). Forty-two, 24, and 28% of the calves were infected with PhA1, PhA6, and Pm, respectively. Ninety-six percent of the calves experienced an increase in anti-PhA1 neuraminidase antibody when sera drawn on feedyard day 28 were compared with sera drawn on the farm. These data demonstrate that the enzyme neuraminidase is produced in vivo in market stressed cattle after a natural Ph infection. Received: 23 March 1998 / Accepted: 4 May 1998