Studies of Antigens for Complement Fixation and Gel Diffusion Tests in the Diagnosis of Infections Caused by Brucella ovis and Other Brucella

Sonically treated and saline-extracted antigens of Brucella ovis, B. canis, B. abortus, and B. melitensis were compared in gel diffusion, complement fixation, and serum absorption tests. All the sonically extracted antigens showed cross-reactions with sera from animals infected or immunized with these species, whereas the saline-extracted antigens were specific for the surface of the rough or smooth colonial phase of the species or strain. The saline-extracted antigens of B. ovis and B. melitensis were both eluted as a single peak in the void volume by Sephadex G-200 column chromatography, in gel diffusion had staining characteristics of lipoproteins, but in immunoelectrophoresis showed distinct mobility patterns. Serological activity for both gel diffusion and complement fixation tests was demonstrated in the immunoglobulin G-containing fraction of sera taken from sheep 12 to 412 days after infection with B. ovis. The gel diffusion test with saline extract of B. ovis is as sensitive as the complement fixation test for the diagnosis of ram epididymitis and is more practical.     

Epididymitis by Brucella ovis: experimental infection in virgin ram lambs.

Ten sexually immature rams were experimentally infected with Brucella ovis, to verify the antibody kinetics and its localization in urinary and genital tracts. All animals became positive to the complement fixation test from the 2nd post infection (p.i.) week and reached the maximum titre (1:256) on the 4th p.i. week. Bacteriemia was demonstrated on 3rd, 4th and 5th p.i. weeks. Two animals, respectively slaughtered 11 and 13 weeks after the infection, showed macroscopic and microscopic genital lesions and the etiologic agent was cultured from their urine and genital organs.     

Preliminary Report on the Development of a Diffusion-in-Gel Method for the Diagnosis of Ram Epididymitis

A simple gel-diffusion technique is described for the diagnosis of ram epididymitis caused by Brucella ovis. The results are shown to be very similar to those obtained by the complement-fixation test, which is currently the standard method of diagnosis. The method is suitable for use in the field and is expected to facilitate the control of ram epididymitis in areas where laboratory facilities are not available.     

An immunoblotting technique for the serodiagnosis of brucellosis by Brucella ovis

An immunoblotting (IB) technique was developed for the serodiagnosis of brucellosis caused by Brucella ovis. Immunoblotting was performed, using a B. ovis HS (hot saline extract) antigen, on 44 blood serum samples which came from rams belonging to known brucella-free flocks, 114 samples originating from ten experimentally B. ovis infected rams and 100 from rams of naturally B. ovis infected flocks. No bands were noted on any of the 44 serum samples which originated from known negative flocks. Sera from naturally and experimentally infected rams identified antibodies to antigenic components with molecular masses of 67, 63, 58, 55, 38, 35, 32, 30, 28, 25, 23, 21, 20-18 (proteins) and 15-12 (RLPS) kDa.     

Evaluation of tests employed in serological diagnosis of brucellosis caused by Brucella ovis

A survey was carried out to verify the sensitivity and specificity of various tests (complement fixation test--CF; agar gel immunodiffusion--AGID; indirect enzyme linked immunosorbent assay--ELISA; immunoblotting--IB) employed in the serological diagnosis of brucellosis caused by Brucella ovis. The tests were executed on 44 blood serum samples of rams coming from B. ovis-free flocks, 75 of B. ovis experimentally infected rams and 1139 from rams living in flocks where B. ovis had been previously isolated. All tests were performed using B. ovis hot saline extract (HS) as antigen. Sensitivity results were 97.4% for IB, 98.68% for CF, 100% for AGID and ELISA; specificity was 100% for all methods. Concordance values were 89.62% (CF-AGID), 78.77% (CF-ELISA), 77.74% (AGID-ELISA), 65.45% (IB-CF), 62.93% (IB-ELISA), 67.24% (IB-AGID). IB identified antibodies to antigenic components with molecular weight of 67, 63, 58, 55, 38, 35, 32, 30, 28, 25, 23, 21, 20-18 kDa (proteins) and 15-12 kDa (rough lipopolysaccharide).     

Sensitive Matrix Gel Diffusion Test for the Detection of Australia Antigen

A matrix gel diffusion (MGD) procedure with a sensitivity comparable to the complement fixation test (CF) has been developed for detecting Australia antigen in serum. The test utilizes a thin layer of agar (0.1 mm) with an applied plastic matrix. Reactants are introduced directly onto the surface of the agar through wells in the plastic matrix. End points obtained by CF with a panel of 11 sera varied from 1:8 to 1:512. When these sera were tested by MGD, end points for detection of antigen were within one dilution of that obtained by CF.     

应用微量玻片凝胶双扩散技术鉴定马铃薯卷叶病毒

应用常规免疫双扩散不能鉴定马铃薯植株中的卷叶病毒(PLRV)。我们用微量玻片凝胶双扩散技术对马铃薯植株汁液中的PLRV进行了鉴定。试验说明,用这种方法可测出马铃薯茎的汁液最高稀释度为1:4,微量玻片凝胶双扩散与普通免疫双扩散灵敏度的比较试验表明,两者可测出提纯PLRV的最低浓度分别为1μg/ml和6μg/ml,但后者不能测出植物汁液中的PLRV。微量玻片凝胶双扩散是目前能够用于检测感病植物汁液中PLRV的最简便易行和可靠的血清学方法。     

Evaluation of Agar-Gel Double Diffusion for the Diagnosis of Adenovirus Infection

A medium is described which can be used to detect starch hydrolysis, gelatinase production, and denitrification by pseudomonads.     

Evaluation of the Interference Filter for Use in Rabies Diagnosis by the Fluorescent Antibody Test

When compared with primary filters widely used for rabies diagnosis by the fluorescent antibody test, an interference filter markedly increased specific staining intensity and contrast.     

Towards the competent conformation for catalysis in the ferredoxin-NADP+ reductase from the Brucella ovis pathogen

Brucella ovis encodes a bacterial subclass 1 ferredoxin-NADP(H) reductase (BoFPR) that, by similarity with other FPRs, is expected either to deliver electrons from NADPH to the redox-based metabolism and/or to oxidize NADPH to regulate the soxRS regulon that protects bacteria against oxidative damage. Such potential roles for the pathogen survival under infection conditions make of interest to understand and to act on the BoFPR mechanism. Here, we investigate the NADP⁺/H interaction and NADPH oxidation by hydride transfer (HT) to BoFPR. Crystal structures of BoFPR in free and in complex with NADP⁺ hardly differ. The latter shows binding of the NADP⁺ adenosine moiety, while its redox-reactive nicotinamide protrudes towards the solvent. Nonetheless, pre-steady-state kinetics show formation of a charge-transfer complex (CTC-1) prior to the hydride transfer, as well as conversion of CTC-1 into a second charge-transfer complex (CTC-2) concomitantly with the HT event. Thus, during catalysis nicotinamide and flavin reacting rings stack. Kinetic data also identify the HT itself as the rate limiting step in the reduction of BoFPR by NADPH, as well as product release limiting the overall reaction. Using all-atom molecular dynamics simulations with a thermal effect approach we are able to visualise a potential transient catalytically competent interaction of the reacting rings. Simulations indicate that the architecture of the FAD folded conformation in BoFPR might be key in catalysis, pointing to its adenine as an element to orient the reactive atoms in conformations competent for HT.     

Analysis of the Crystal Antigens of Bacillus thuringiensis by Gel Diffusion

S ummary . The antigenic patterns of the crystal protein inclusions of Bacillus thuringiensis were determined. No specific antigenic patterns associated with previously described subgroups of this species were found. A larger number of categories of crystal antigen than of flagellar antigen or esterase type were found. In some cases isolates indistinguishable in classifications based on flagellar antigens or esterase types could be subdivided by their crystal antigenic pattern. The use of crystal antigens in classification is discussed.     

Extraction of membrane antigens from Brucella ovis and an assessment of their serological activity by immunoblotting

The efficacy and selectivity of chaotropic and phase-partitioning procedures for the extraction of membrane proteins from Brucella ovis were compared with a standard Sarkosyl method. Major group 1, 2 and 3 outer-membrane proteins (OMPs) of B. ovis stained by Coomassie blue in SDS-PAGE gels had, respectively, apparent molecular masses of 81/82 kDa, 39-41 kDa and 30-32 kDa. The presence of these bands in the Sarkosyl extract of total membrane vesicles (TMVs) indicate that the procedure failed to selectively solubilize only inner-membrane proteins (IMPs). SDS-PAGE analyses also revealed the presence of OMPs and other additional bands following extraction of B. ovis TMVs by butanol phase-partitioning or with extraction solutions based on the chaotropic reagents potassium thiocyanate (KSCN), sodium salicylate (SSC) and lithium acetate (LAE). OMPs are therefore not selectively extracted by any one of these procedures. Based on the number and staining intensity of extracted membrane-associated polypeptides, the efficacy of different extraction procedures could be graded in decreasing order as follows: KSCN, SSC, butanol and LAE. Both butanol and SSC were particularly effective in extracting group 3 OMPs. Sera from chronic excretor rams were used to identify zones of seroreactivity in immunoblots. Essentially, two reactivity patterns were seen: strong antibody binding against polypeptides in zones A (46-85 kDa), C (28-32 kDa) and D (18-22 kDa) in one, and additional reactivity against zones B (34-44 kDa) and E (13-18 kDa) polypeptides in the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of agar gel precipitation and complement fixation test for the detection of Brucella ovis infection.

Blood samples from 710 sheep raised in 20 different farms were examined for Brucella ovis antibodies. Thirty samples were positive with complement fixation, 35 samples with precipitation, and 58 samples with both tests. It has been concluded that the gel precipitation technique is adequate for screening but for individual testing both methods should be used simultaneously.     

Evaluation of Rapid IgG4 Test for Diagnosis of Gnathostomiasis

Human gnathostomiasis is a parasitic disease caused by Gnathostoma nematode infection. A rapid, reliable, and practical immunoassay, named dot immuno-gold filtration assay (DIGFA), was developed to supporting clinical diagnosis of gnathostomiasis. The practical tool detected anti-Gnathostoma-specific IgG4 in human serum using crude extract of third-stage larvae as antigen. The result of the test was shown by anti-human IgG4 monoclonal antibody conjugated colloidal gold. The sensitivity and specificity of the test were both 100% for detection in human sera from patients with gnathostomiasis (13/13) and from healthy negative controls (50/50), respectively. Cross-reactivity with heterogonous serum samples from patients with other helminthiases ranged from 0 (trichinosis, paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis) to 25.0% (sparganosis), with an average of 6.3% (7/112). Moreover, specific IgG4 antibodies diminished at 6 months after treatment. This study showed that DIGFA for the detection of specific IgG4 in human sera could be a promising tool for the diagnosis of gnathostomiasis and useful for evaluating therapeutic effects.     

Gel Diffusion Method for Determining the Titer of Duck Hepatititis Virus

Apparatus necessary for a quantitative gel diffusion and precipitation assay of duck hepatitis virus was designed and constructed.     

Field Evaluation of a Coproantigen Detection Test for Fascioliasis Diagnosis and Surveillance in Human Hyperendemic Areas of Andean Countries

Background

Emergence of human fascioliasis prompted a worldwide control initiative including a pilot study in a few countries. Two hyperendemic areas were chosen: Huacullani, Northern Altiplano, Bolivia, representing the Altiplanic transmission pattern with high prevalences and intensities; Cajamarca valley, Peru, representing the valley pattern with high prevalences but low intensities. Coprological sample collection, transport and study procedures were analyzed to improve individual diagnosis and subsequent treatments and surveillance activities. Therefore, a coproantigen-detection technique (MM3-COPRO ELISA) was evaluated, using classical techniques for egg detection for comparison.

Methodology and Findings

A total of 436 and 362 stool samples from schoolchildren of Huacullani and Cajamarca, respectively, were used. Positive samples from Huacullani were 24.77% using the MM3-COPRO technique, and 21.56% using Kato-Katz. Positive samples from Cajamarca were 11.05% using MM3-COPRO, and 5.24% using rapid sedimentation and Kato-Katz. In Huacullani, using Kato-Katz as gold standard, sensitivity and specificity were 94.68% and 98.48%, respectively, and using Kato-Katz and COPRO-ELISA test together, they were 95.68% and 100%. In Cajamarca, using rapid sedimentation and Kato-Katz together, results were 94.73% and 93.58%, and using rapid sedimentation, Kato-Katz and copro-ELISA together, they were 97.56% and 100%, respectively. There was no correlation between coproantigen detection by optical density (OD) and infection intensity by eggs per gram of feces (epg) in Cajamarca low burden cases (<400 epg), nor in Huacullani high burden cases (≥400 epg), although there was in Huacullani low burden cases (<400 epg). Six cases of egg emission appeared negative by MM3-COPRO, including one with a high egg count (1248 epg).

Conclusions

The coproantigen-detection test allows for high sensitivity and specificity, fast large mass screening capacity, detection in the chronic phase, early detection of treatment failure or reinfection in post-treated subjects, and usefulness in surveillance programs. However, this technique falls short when evaluating the fluke burden on its own.     

Evaluation of the Corneal Test as a Laboratory Method for Rabies Diagnosis

The corneal test (CT) for rabies diagnosis was evaluated in samples from 313 subjects of different species. Some of the subjects were inoculated experimentally and others were naturally infected. When the CT was compared with immunofluorescence staining and mouse inoculation tests on brains of the same subjects, a sensitivity of 41.7% and a specificity of 100% were found. The authors conclude that a positive CT result would confirm the diagnosis of rabies, but a negative one would not exclude the possibility of disease.     

Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.