Serogroup-specific Antibodies Research Articles

Development of a competitive ELISA based on the LSDV A33 antigen

Goatpoxvirus (GTPV), sheeppoxvius (SPPV), and the Lumpy skin disease virus (LSDV) is a Capripoxvirus belonging to the family poxviridae. They can cause significant economic losses in countries where this disease are endemic. However, effective and convenient diagnostic tools against sera antibody are not readily available until now. Toward this goal, a polyclonal antibody competitive enzyme-linked immunosorbent assay (c-ELISA) of detecting serogroup-specific antibody is established based on major LSDV antigen A33. Serum samples (n = 605) were collected to optimize the c-ELISA from different areas. The cut-off value for the c-ELISA was estimate using percent inhibition (PI) values. The diagnostic performance of test including sensitivity (sn) and specificity (sp) were obtained by receiver operator characteristic (ROC) analysis. Among these analysis, > 57.61% PI value was accepted as cut-off of the c-ELISA, the diagnostic sn an diagnostic sp were reached to 96.4% and 98.5%, at > 95% confidence interval. These results show that the developed competitive ELISA is sensitive, specific, and reliable, which make it appropriate for serological investigation.

Antigenic determinants driving serogroup-specific antibody response to Neisseria meningitidis C, W, and Y capsular polysaccharides: Insights for rational vaccine design

Meningococcal glycoconjugate vaccines sourced from capsular polysaccharides (CPSs) of pathogenic Neisseria meningitidis strains are well-established measures to prevent meningococcal disease. However, the exact structural factors responsible for antibody recognition are not known.CPSs of Neisseria meningitidis serogroups Y and W differ by a single stereochemical center, yet they evoke specific immune responses. Herein, we developed specific monoclonal antibodies (mAbs) targeting serogroups C, Y, and W and evaluated their ability to kill bacteria. We then used these mAbs to dissect structural elements responsible for carbohydrate-protein interactions. First, Men oligosaccharides were screened against the mAbs using ELISA to select putative lengths representing the minimal antigenic determinant. Next, molecular interaction features between the mAbs and serogroup-specific sugar fragments were elucidated using STD-NMR. Moreover, X-ray diffraction data with the anti-MenW CPS mAb enabled the elucidation of the sugar-antibody binding mode.Our findings revealed common traits in the epitopes of all three sialylated serogroups. The minimal binding epitopes typically comprise five to six repeating units. Moreover, the O-acetylation of the neuraminic acid moieties was fundamental for mAb binding.These insights hold promise for the rational design of optimized meningococcal oligosaccharides, opening new avenues for novel production methods, including chemical or enzymatic approaches.

Serological and molecular detection as well as typing of Leptospira spp. in foxes, raccoons, and other wild carnivores in North-Eastern Germany, 2021–2022

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. While the latter are reported from various mammal hosts such as humans, dogs, or rodents, less is known about their presence in wild carnivores. We therefore investigated the presence of Leptospira spp. in foxes, raccoons, badgers, raccoon dogs, and martens in North-Eastern Germany. Kidney, urine, and blood specimens obtained from legally hunted or road-killed animals were tested by real-time PCR and by serogroup specific antibody detection for the presence of Leptospira spp. Additionally, kidney and urine specimens were tested by real-time PCR for the presence of Brucella spp. and Francisella tularensis, with all being negative for these two zoonotic pathogens. Leptospira spp. were detected by PCR in 12.6 % (n = 21/166) and serologically in 26.2 % (n = 53/202) of tissue and serum samples, respectively. Antibodies to 15 different serogroups were identified with Javanica (n = 25) and Bataviae (n = 12) being predominant. A high sero-prevalence of 34.0 % and 18.6 % in foxes and raccoons, respectively, and the presence of ST17 associated with human and animal leptospirosis indicates a reservoir and the zoonotic potential of these wild animals.

Changing Trend in the Prevalence and Emergence of Leptospira Serogroup-Specific Antibodies in Livestock in Gujarat, India

The study was undertaken to investigate the frequency distribution of predominant Leptospira serovars as well as to establish the prevalence of Leptospira antibodies in enzootic Surat and Navsari districts of south Gujarat. A total of 1014 purposive random serum samples from livestock species (Cattle n = 622; Buffaloes n = 246 and Goats n = 146) were collected in the rainy season during 2015–2016. The samples were tested in microscopic agglutination test using a panel of 18 reference Leptospira serovars covering 16 Leptospira serogroups. The overall seroprevalence of 30.6% (310/1014) with 25.9% in cattle, 39.0% in buffaloes and 36.3% in goats were observed in the study. Further, the Chi-squared test analysis showed that the presence of antibodies was associated with the studied districts (χ2 = 123.7, p < 0.05) and age group of livestock species (Bovine-χ2 = 15.97, p < 0.05). Out of 310 reacted sera, 151 samples showed reactivity with more than one serovars representing 14. 9% prevalence of multiple serovars among the livestock species. Moreover, the frequency distribution of predominant Leptospira antibodies was determined against the employed serovars: Panama—25.5%, Hebdomadis—23.9%, Javanica—22.3%, Tarassovi—21.6%, Hurstbridge—21.6%, Bataviae—21.3%, Pyrogenes—20.7%, Pomona—19.4%, Icterohaemorrhagiae—18.7%, Grippotyphosa—17.7%, Hardjo—17.42%, etc. The present study significantly provides the predominant Leptospira serovars and its frequency distribution in livestock, which had a role in maintaining various serovars with the change in the trend over a period of years in south Gujarat. These findings would also help in strategizing and mitigating the leptospirosis burden in enzootic regions.

Shiga toxin-producing Escherichia coli in household members of children with hemolytic uremic syndrome.

The objective is to establish the frequency of STEC infections in household contacts of HUS patients. We studied 292 household contacts of 82 HUS patients attended from 2010 to 2018. In HUS cases, diagnostic criteria were (1) isolation and characterization of STEC strains, (2) detection of free fecal Shiga toxin (FFStx), and (3) detection of anti-O serogroup-specific antibodies. Contacts were studied by screening of stx genes by polymerase chain reaction and/or STEC isolation from stool samples. Clonal relation of STEC strains was established by pulsed-field gel electrophoresis (PFGE). Frequencies of HUS patients without STEC isolation with STEC-positive contacts were determined. Serotypes and stx-genotypes in patients and contacts were analyzed. Thirty (36.6%) HUS patients had 36 STEC-positive contacts. Fourteen (38.8%) were children, 20 adults, and 2 dogs. One sibling developed HUS, 6 contacts had gastrointestinal symptoms, and the rest were asymptomatic. In 5 of 30 HUS patients, STEC infection could not be confirmed, and 2 cases were diagnosed only by FFStx detection. Of the remaining 23 HUS patients, 16 had E. coli O157 and 7 E. coli O145 infection. Serotype and/or stx-genotype concordance was established in 19 (83%) of 23 HUS patients and their contacts. Five HUS cases and their contacts studied by PFGE showed macrorestriction patterns with more than 90% similarity. Nearly one third of HUS patients had STEC-positive family contacts, and one third of them were children. Early identification is important to prevent ongoing contamination among family and institutional contacts and to facilitate prompt detection of HUS in STEC-positive contacts.

A multi-species indirect ELISA for detection group-specific antibodies against VP7 protein of bluetongue virus

Abstract In the present study, a multispecies indirect ELISA for the detection of serogroup-specific antibodies against the VP7 protein of bluetongue virus (BTV) has been developed. The assay was optimized using a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Serum samples (n = 515) from small and large domestic ruminants were used to optimize the assay. The percent positivity (PP) values were used for the estimation of cut-off values. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >20.24% PP value was accepted as the cut-off at which DSn was 96.05% with 92.9–98.1 (95%) confidence interval and DSp was 98.47 with 96.1–99.6 (95%) confidence interval. The above results indicate that the multispecies i-ELISA assay is sensitive, specific and minimize the dependence of species-specific conjugated antibodies for serological investigation of bluetongue in small and large ruminants.

Seroprevalence and distribution of serogroup-specific pathogenic Leptospira antibodies in cattle and buffaloes in the state of Andhra Pradesh, India.

Aim:In this study, the prevalence and the distribution status of Leptospira serogroup-specific antibodies among cattle and buffaloes in enzootic districts of Andhra Pradesh, a South Indian state was carried out.Materials and Methods:A total of 426 serum samples were randomly sampled from various villages from Prakasam, Kurnool, Guntur, Chittoor, Srikakulam, Visakhapatnam, and Godavari districts of Andhra Pradesh between 2016 and 2017. Serum samples from cattle (n=106) and buffaloes (n=320) having a history of pyrexia, and reproductive problems such as agalactia, infertility, abortions, and stillbirth. The serum samples were screened for Leptospira-specific antibodies by microscopic agglutination test using a reference panel of 18 live cultures of pathogenic Leptospira serovars.Results:The overall seropositivity of 68.08% (290/426) was observed with 70.8% (75/106) in cattle and 67.18% (215/320) in buffaloes. The frequency distribution of predominant serogroup-specific Leptospira antibodies in the sampled areas was determined against the employed serovars as follows: Icterohaemorrhagiae – 21.38%, Hebdomadis – 18.97%, Australis – 18.62%, Pomona – 17.24%, Sejroe – 15.86%, Tarassovi – 15.86%, Autumnalis – 15.52%, Panama – 14.83%, Shermani – 12.07%, Javanica – 11.38%, Hurstbridge – 11.03%, and Pyrogenes – 10.69%.Conclusion:It was evident that bovines had a role in maintaining several predominant Leptospira serovars with the change in the trend over a period. The results from this study would also help in strategizing and mitigating the disease burden in cattle and buffaloes of the enzootic area.

Prevalence of Leptospira serogroup-specific antibodies in cattle associated with reproductive problems in endemic states of India.

In this study, the seroprevalence and distribution of Leptospira in dairy cattle in endemic states of India were investigated in association with reproductive problems of the cattle. A total of 373 cattle serum samples from 45 farms in Maharashtra, Gujarat, Andhra Pradesh, Telangana, Karnataka, Tamil Nadu, Punjab, Haryana, Chhattisgarh, Sikkim and Uttarakhand states were collected from animals with a history of reproductive disorders like abortion, repeat breeding, anoestrus and endometritis, and also from apparently healthy animals. These samples were screened for Leptospira serogroup-specific antibodies by microscopic agglutination test (MAT) using a panel of 18 live reference serovar antigens. The seropositivity of 70.51% (263/373, 95% CI 0.65 to 0.75) was associated with reproductive problems (χ2 = 55.71, p < 0.01) and sampled states (χ2 = 32.99, p < 0.01) and independent of apparently healthy animals (χ2 = 15.6, p > 0.10) and age groups of cattle (χ2 = 0.91, p > 0.10). Further, the odds (risk-relation) of reproductive disorders was 5.29 compared to apparently healthy animals (0.25 odds). The frequency distribution of predominant serogroup-specific Leptospira antibodies were determined against the serovars: Hardjo (27.76%), Pyrogenes (18.63%), Canicola and Javanica (17.49%), Hebdomadis (17.11%), Shermani and Panama (16.73%), Djasiman (16.35%), Tarassovi, Grippotyphosa and Pomona (15.97%), Icterohaemorrhagiae (15.59%), Copenhageni (14.83%), Australis (13.69%), Kaup and Hurstbridge (10.65%), Bankinang (10.27%) and Bataviae (9.51%). In conclusion, dairy cattle have a role in maintaining important several serovars besides well-known Hardjo serovar in endemic states of India and warrant mitigating measures to reduce the incidence of cattle leptospirosis including need for an intensive surveillance programme, preventive vaccination and control strategies.

Distribution of serogroup specific antibodies against leptospirosis in livestock in Odisha

The study was conducted to identify epidemiological sero-distribution of Leptospira serovars representing serogroups of antibodies in Odisha, Eastern part of India. Samples were collected from livestock during 2011-2014. A total of 537 non-purposive random serum samples from 12 districts in Odisha were tested at 1:100 dilutions in microscopic agglutination test (MAT) using live antigens of 18 reference Leptospira serovars. The overall prevalence of 36.69% (197/537) with 36.13% in cattle, 54.28% in buffaloes, 28.33% in goats and 44.44% in sheep was observed. Out of 197 reacted sera, 100 samples showed reactivity with more than one serovars representing 50.76% prevalence of multiple serovars. On analysis, the seroprevalence of leptospirosis in cattle (·2 = 63.41, P&lt;0.01), buffaloes (·2 = 14.68, P &lt;0.01), sheep (·2 = 54.66, P &lt;0.01) and goats (·2 = 36.27, P &lt;0.01) across different regions are significantly dependent. The predominant Leptospira serogroup specific antibodies against major serovars representing Hardjo (30.4%), Tarassovi (20.8%), Australis (19.19%), Bankinang (18.18%), Pomona (16.66%), Kaup (15.65%), Hebdomadis (11.11%), Pyrogenes (10.1%), Bataviae (9.59%), Icterohaemorrhagiae (9.09%), Shermani (7.57%), Djasiman/Javanica (6.56%), Hurstbridge (5.55%), Grippotyphosa (4.54%), Panama (4.04%), Canicola (3.03%) and Copenhageni (2.02%) against frequency distribution were noticed. The overall prevalence of antibodies detected against the serovars Hardjo, Tarassovi, Australis, Bankinang, Pomona, Kaup, Hebdomadis and Hurstbridge representing specific serogroups suggested that these serovars may be of use in the reference panels of Leptospira antigen in the MAT for this region in both human and animal state or district disease diagnostic laboratories.

A competitive ELISA for detection of group specific antibody to bluetongue virus using anti-core antibody

Bluetongue virus (BTV) is transmitted by biting midges, which infects domestic and wild ruminants. In present study, a competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of serogroup-specific antibodies against VP7 protein of BTV has been developed. The assay measures the competition between a group specific antibody against core protein of BTV and a test serum to an optimized concentration of BTV recombinant-VP7 (r-VP7) antigen. Serum samples (n = 895) collected from small and large ruminants were used to optimize the C-ELISA. Percent inhibition (PI) values were used for estimation of the cut-off value for the C-ELISA. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >50% PI value was accepted as cut-off at which DSn and Dsp was achieved as 97.6% and 98.0% respectively, at >95% confidence interval. Results show the present C-ELISA assay described to be sensitive, specific and reliable and could be adopted for serological investigation of small and large ruminants.

Evaluation of an indirect ELISA for detection of antibodies in bulk milk against bluetongue virus infections in the Netherlands

After the introduction of bluetongue virus serotype 8 (BTV-8) in western Europe in 2006, an indirect ELISA for detection of serogroup-specific antibodies against BTV in serum samples was validated for individual milk samples by the Central Veterinary Institute and the Animal Health Service in the Netherlands ( Kramps et al., 2008). In order to develop a cost-effective monitoring tool, we now have evaluated this ELISA also for use in bulk milk. Therefore, bulk milk samples and individual milk samples were collected from 92 herds in the affected southern region in the Netherlands in 2007, before the start of the vaccination campaign. In addition, bulk milk samples collected from 88 herds before the bluetongue introduction in 2006 (“historically negative” samples) have been tested. With these results ROC analyses were performed and herd specificity and herd sensitivity of the bulk milk ELISA were estimated. All “historically negative” bulk milk samples were negative in the ELISA, with a mean S/ P ratio of 10 ± 0.8%. The herd sensitivity and herd specificity of the ELISA in bulk milk samples depend on the cut-off that is chosen. In order to detect a within-herd-prevalence of 1%, the optimal cut-off S/ P ratio 13% was found. A few herds with one or two milk-positive animals would then be missed. The specificity will be 100%. A within-herd-prevalence of 10% can be detected with 100% sensitivity at a cut-off S/ P ratio of 96%. In conclusion, the indirect ELISA in bulk milk samples is a very specific and sensitive test which can be implemented in monitoring and surveillance systems in unvaccinated populations.

Development and evaluation of a rapid multianalyte particle-based flow cytometric assay for the quantification of meningococcal serogroup B-specific IgM antibodies in sera for nonculture case confirmation

Serogroup-specific antibody has been shown to be present in the sera of patients recovering from meningococcal disease, and thus the detection of such antibodies may aid in the confirmation of disease. There are currently no standard methods for measuring meningococcal serogroup B-specific antibody in sera. Here, we report the development of a microsphere-based immunoassay which utilizes colominic acid from Escherichia coli 07:K1 (L):NM to detect immunoglobulin M directed against serogroup B polysaccharide. The serogroup B assay was incorporated into a multiplex assay which also detects serogroup-specific immunoglobulin M for meningococcal serogroups A, C, Y and W-135. Using the method of cross-standardization, serogroup B-specific immunoglobulin M concentrations were assigned to the standard serum CDC 1992. The assay is able to detect increases in specific immunoglobulin M concentrations from acute to convalescent phase serum from serogroup B cases, and can be utilized in conjunction with the previously developed tetraplex immunoglobulin G detection assay for serogroups A, C, Y and W-135.

Induction of immunological memory in UK infants by a meningococcal A/C conjugate vaccine.

The induction of immunological memory to serogroup A and C polysaccharides in UK infants immunized with three doses of a meningococcal A/C oligosaccharide CRM197 conjugate vaccine was investigated. Forty UK infants vaccinated previously with three doses of a meningococcal A/C oligosaccharide-CRM197 conjugate vaccine at 2, 3 and 4 months of age, were revaccinated at a mean age of 145.6 weeks with either a 10 or 50 microg dose of licensed meningococcal A/C polysaccharide vaccine. Serogroup-specific antibody and serum bactericidal antibody (SBA) responses were measured by enzyme-linked immunosorbent assay and serum bactericidal assays, respectively. Following challenge, anti-serogroup A and C polysaccharide antibody levels rose from pre-booster geometric mean concentrations (GMC) of 3.1 and 2.1 microg/ml respectively to 19.6 and 21.0 microg/ml 1 month post-booster. Serum bactericidal antibody geometric mean titres (GMTs) for serogroups A and C increased 156- and 113-fold from 2.1 and 7.1 pre-booster respectively to 327.4 and 800.7 post-booster. A serogroup A control group of 45 children received a 10 microg dose of licensed meningococcal A/C polysaccharide vaccine (with no prior history of serogroup A vaccination) had serogroup A SBA GMTs of 2.3 pre-vaccination rising to 8 post-vaccination with corresponding GMCs of 0.8 and 10.8 microg/ml. These rises in SBA following serogroup A/C conjugate vaccination are indicative of immunological priming.

Immune response to non-O157 Vero toxin-producing Escherichia coli in patients with hemolytic uremic syndrome.

The incidence and time course of the immune response to Vero toxin-producing Escherichia coli (VTEC) other than O157 (O26, O55, O111, O128) were determined in 107 children with enteropathic hemolytic uremic syndrome (HUS) by ELISA and compared to antibody profiles of 125 healthy pediatric controls and 100 children with community-acquired uncomplicated diarrhea. Six of 8 HUS patients with non-O157 VTEC isolates exhibited a serologic response to the homologous lipopolysaccharide antigen similar to that of patients infected with E. coli O157. In addition, elevated IgM or IgG antibodies were demonstrated in 7 of 19 culture- and O157 serology-negative HUS patients. Serogroup-specific antibodies decreased below cutoff levels within several months after convalescence. Anti-non-O157 antibodies were found in 14% of the diarrheic controls. Unexpectedly, a high proportion (27/82) of anti-O157 lipopolysaccharide antibody-positive HUS samples reacted with LPS from E. coli O55:B5, suggesting shared epitopes with endemic VTEC O157 strains.

Evaluation of a competitive enzyme-linked immunosorbent assay to detect infection of cattle in sentinel herds in Queensland, Australia with bluetongue viruses

A competitive enzyme-linked immunosorbent assay (ELISA) was used to detect serogroup specific antibodies to bluetongue viruses. This test is commercially available and was evaluated with serially collected sera from 10 sentinel herds of cattle maintained in Queensland, Australia during 1994. Determination of an infection during the period of observation was based on the development of a serum neutralisation (SN) test titre ≥ 1:8 to any one of 8 bluetongue virus serotypes known to exist in Australia. Using the inhibition value of 40% recommended by the manufacturer to classify cattle as exposed to bluetongue viruses, the ELISA was highly sensitive (100%; 95% confidence interval, 77.9–100%) and moderately specific (86.4%; 95% CI, 77.0–93.0%), relative to the SN test. An inhibition value of 70% maximised both sensitivity (100%, lower 95% CI, 77.9%) and specificity (93.2%, 95% CI, 85.2–98.0%) of the ELISA. The chance (posttest probability) of an animal from which a serum sample had an inhibition value ≥ 70% in the ELISA developing an SN test titre ≥ 1:8 was 93.6% (95% CI, 86.4–97.9%). Investigations of the temporal development of ELISA and SN test reactions, showed that the ELISA detected exposure to bluetongue viruses significantly ( P = 0.0156) earlier than the SN test. The bluetongue virus ELISA is a useful test in surveillance programs. False positive assay results make it inappropriate for monitoring and diagnosis, unless it is used in conjunction with the SN test.

Manipulation of the helper T cell response to influence antigenic competition occurring with a multivalent vaccine.

The reduction in antibody observed following inoculation with multiple heterologous Dichelobacter nodosus pili antigens is thought to be due to competition between antigen-specific B cells for a limited amount of T cell help. We demonstrate here that this competition is not further influenced by the expansion of cross-reactive antibody secreting cells at the expense of serogroup specific antibody secreting cells. The T cell determinants of pili recognized by sheep and BALB/c mice have been defined using 15 residue peptides. These T cell determinants include cross-reactive determinants in the conserved amino terminal region of the antigen. Here we investigate the effect of expanding the pili-specific T cell population by priming with pili derived T cell determinants. It was not possible to increase the antibody elicited in response to the multivalent vaccine by priming mice with either a synthetic peptide spanning a T cell determinant or with reduced and alkylated or heterologous serogroups of pili 4 weeks before inoculation with the multivalent vaccine. A strategy designed to increase the T cell population by inoculating animals with pili covalently coupled to an extrinsic T cell determinant was pursued.

Immunological parameters associated with antigenic competition in a multivalent footrot vaccine

A murine model for antigenic competition with multivalent D. nodosus pili vaccine has been established that parallels the phenomenon observed in sheep where levels of antibody, specific for any particular serogroup of pili, are significantly lower following vaccination in the presence of multiple serogroups of pili than with that serogroup alone. This competition was observed in both high and low responder strains of mice and was not dependent on the multiplicity of the antigens in the multivalent vaccine but could be observed with a large excess of a single heterologous serogroup. Competition was manifest by a reduction in the number of serogroup-specific antibody secreting cells elicited in response to vaccination. The antibody response to a single serogroup of pili reached a plateau at high doses and it was at these doses that antigenic competition was most pronounced, under conditions where both B- and T-cell responses were limiting. The limit in T-cell responsiveness was not imposed at the level of presentation of antigen. Pili-specific T cells were largely cross-reactive for different serogroups, and under conditions of limiting T-cell stimulation within a lymph node the available T cells would have to be shared between B cells specific for each serogroup of pili, which may in turn result in the decrease of serogroup-specific antibody induced following inoculation with the multivalent vaccine.

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits ofLeptospira interrogansserovarpomona

Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona, were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona. In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.

Occurrence and nature of mixed infections in nodules of field-grown soybeans (Glycine max)

Mixed infections of Bradyrhizobium japonicum strains in early and late nodules of four soybean cultivars were studied in a field soil. Nodule occupants were identified by immunofluorescence using serogroup specific antibodies prepared against B. japonicum strains USDA 110, USDA 123, and USDA 138. Double infection was determined directly by combined examination of the same microscopic field by fluorescence and phase contrast microscopy. Double strain occupancy was observed consistently, and its occurrence did not differ substantially in pouch, soil pot, and field experiments, ranging in incidence from 12% to 32%. No significant differences in the incidence or nature of double infection could be attributed to cultivar, seed inoculation, or plant maturity. Strains reactive to strain USDA 123-fluorescent antibody were dominant in both singly and doubly infected nodules irrespective of cultivar, plant age, or seed inoculation with strain USDA 110.

Rapid diagnosis of respiratory pseudomonas infections--indirect immunofluorescent-staining of sputum with monoclonal antibody to Pseudomonas aeruginosa

For the rapid diagnosis of respiratory infections caused by Pseudomonas aeruginosa, we developed IFA-staining of sputum for Pseudomonas aeruginosa using serogroup-specific monoclonal antibody (MEIASSEI RYOKUNOUKIN, MEIJI, JAPAN).IFA-titers of each serogroup-specific antibodies were distributed from 1: 4 to 1: 200 dillution, and minimal concentration of bacteria detectable by IFA-staining was 106 CFU/ml in the sputum. We performed IFA-staining on 131 smears of the sputa presented to the Clinical Laboratory of Nagasaki University. In 36 specimens from which more than 106 CFU/ml of Pseudomonas aeruginosa were isolated, 27 specimens (75%) were judged as IFA-positive, and in 18 specimens with more than 107 CFU/ml of Pseudomonas aeruginosa, 15 (83%) were IFA-positive. Furthermore, there was a distinct correlation between number of fluorescing bacilli (microscopic findings of IFA-staining) and quantity of Pseudomonas aeruginosa isolated from the sputum. In the 86 sputa with a negative culture for Pseudomonas aeruginoas, 82 (95%) were judged as IFA-negative.Using this method, we can directry identify Pseudomonas aeruginosa in the sputum, and according to these results, the IFA-staining of the sputum was considered to be a useful method for the rapid diagnosis of respiratory infections caused by Pseudomonas aeruginosa.