anti-H Serum Research Articles

Molecular genotyping of Escherichia coli o111 isolated from children with diarrhea in rural and urban areas

Introduction: Enteropathogenic Escherichia coli (EPEC) is a group of bacteria associated with infantile diarrhea. EPEC is defined as being typical (tEPEC) or atypical (aEPEC) depending on whether it presents the EPEC adherence factor (EAF) plasmid or not; tEPEC presents EAF. Objective. Carry out a retrospective study of E. coli strains isolated from children with diarrhea in both rural and urban areas to determine if they belong to tEPEC or aEPEC groups. Material & Methods: Strains that had been reported to be E. coli were analyzed by PCR using specific primers and 188 anti-O sera and 53 anti-H sera. The presence of eae, bfp, eafA, perA, espC and espP genes associated with virulence was analyzed, as was the presence of arpA, chuA, yjaA and TspE4 to define phylogroups. Results: Fifty-three cultures of E. coli isolated between 1985 and 2003 from 35 children under five years of age with severe diarrhea were analyzed. Twelve (34.3%) children came from a rural area and 23 (65.7%) from an urban area. Serologic typing identified the following serotypes: O111ab:NM (67.9%), O111ab:H2 (20.8%), O111ab:H12 (9.4%) and O111ac:H8 (1.9%). The presence of eae was positive in all 53 strains, while bfp and perA was detected in 38 (71.7%) strains and eaf in 35 (66%) strains. The combination of eae/bfp used to determine typical strains was positive in 38 (71.7%) strains and negative in 15 (28.3%) strains. Analysis by year and region showed that between 1985 and 1987, 35 (66%) strains were typical and 4 (7%) atypical in the rural area and urban area1. The same analysis between 1994 and 2003 in the urban areas showed that 11 (20.8%) strains were atypical, which were eae positive but eaf/bfp/perA negative, and 3 (5.7%) strains were typical. Phylogenetic grouping identified B1 and A in 38 (71.7%) and 9 (17%) strains respectively, and 2 (3.8%) each of B2, D and F. Conclusion. Typical EPEC strains were more prevalent in both the rural and urban areas between 1985 and 1987. In contrast, the presence of atypical EPEC strains was more prevalent in urban areas in more recent years. Analysis showed that the strains fell predominantly into commensal phylogroups.

Diversity of Potentially Pathogenic Escherichia coli O104 and O9 Serogroups Isolated before 2011 from Fecal Samples from Children from Different Geographic Regions.

In 2011, an outbreak of hemorrhagic colitis and hemolytic uremic syndrome (HUS) was reported in Europe that was related to a hybrid STEAEC of Escherichia coli (E. coli) O104:H4 strain. The current study aimed to analyze strains of E. coli O104 and O9 isolated before 2011. The study included 47 strains isolated from children with and without diarrhea between 1986 and 2009 from different geographic regions, as well as seven reference strains. Serotyping was carried out on 188 anti-O and 53 anti-H sera. PCR was used to identify DEC genes and phylogenetic groups. Resistance profiles to antimicrobials were determined by diffusion in agar, while PFGE was used to analyze genomic similarity. Five serotypes of E. coli O104 and nine of O9 were identified, as well as an antigenic cross-reaction with one anti-E. coli O9 serum. E. coli O104 and O9 presented diarrheagenic E. coli (DEC) genes in different combinations and were located in commensal phylogenetic groups with different antimicrobial resistance. PFGE showed that O104:H4 and O9:(H4, NM) strains from SSI, Bangladesh and México belong to a diverse group located in the same subgroup. E. coli O104 and O9 were classified as commensal strains containing DEC genes. The groups were genetically diverse with pathogenic potential making continued epidemiologic surveillance important.

Serotypes, virulence genes profiles and antimicrobial resistance patterns of Escherichia coli recovered from feces of healthy lambs in Mexico

Healthy lambs are one of the major reservoirs of Shiga toxin-producing Escherichia coli (STEC) and it is known as the cause of foodborne diseases (FBD). The work objective is to characterize (STEC) isolates obtained from rectal swabs of healthy lambs herds, a total of 183 samples were obtained from sheep production units of the State of Mexico. E. coli isolates were confirmed through the amplification of the uid A gene. antimicrobial sensitivity pattern was determined through Kirby-Bauer (CLSI, 2012) test and the presence stx1, stx2 and eae genes from isolates by multiplex PCR. Serotyping was performed using specific anti-O and anti-H sera (SERUNAM, Mexico) for 185 Somatic and 56 flagellar antigens. 126 isolates biochemically and molecularly identified as E. coli were obtained, of which 80 did not express any virulence factor and 46 expressed at least some (STEC) virulence factor. The highest percentage of E. coli resistance was for tetracycline 48.7% (39/80), followed by nalidixic acid 13.7% (11/80), gentamicin 6.2% (5/80) and Ciprofloxacin 3.7% (3/80). Resistance to amikacin, cefotaxime and ceftazidime were not detected. A frequency of 46 STEC isolates (36.2%) were obtained, of which 28/46 (22.0%) expressed stx1, stx2 3/46 (6.5%), stx1, stx2 13/46 (10.2%) and eae 2/46 (1.6%). Thirty different serotypes were obtained. The three serotypes with the highest number of isolates (four each) were: O76:H19, O118:H27 and O146:H21 which have been identified as a cause of diarrhea in human population. An isolate of serogroup O104 was obtained, with a significant importance for European public health. In virtue of the discovered serotypes and the virulence factors distribution, we can affirm that the obtained isolates from lambs in the State of Mexico are classifiable as atypical STEC of low virulence.

Diminished expression of B antigen mimicking B3 phenotype in a patient with AML-M3: a rare case report.

The ABO blood group system plays an important role in transfusion and transplantation practices. Correct identification of the blood group of an individual is of paramount importance as minor errors can lead to fatal transfusion reactions. Weak expression of A, B and H antigens on the red cell surface can be inherited or acquired. They are probably the least frequently encountered and pose a challenge in the routine immunohematology practice.1 Such antigenic changes in hematological malignancies most commonly involve the ABO blood group system.2 The strength of agglutination with anti-B, anti-A,B, and anti-H serum, presence or absence of ABO isoagglutinins in the serum, adsorption-elution studies and the presence or absence of A, B and H substance in saliva along with pedigree analysis help to characterize the weaker variants of A and B serologically. Possible phenotypic subgroups include A3, Aend, Ax, Am, Ay, and Ael. Subgroups of B are very rare, less frequent than those of A and are classified as B3, BX, Bm and Bel in decreasing order of the amount of the B antigen (Table 1).

Évaluation du groupage sanguin dans les systèmes ABO et Rh dans les formations sanitaires du Bénin

Study purposeThe goal of this work is to assess the modalities of blood typing achievement in Benin with the view of their improvement. MethodsOn the basis of a questionnaire including the detailed operative process, a prospective investigation has been achieved in public and private health centers laboratories. ResultsIt came out that the execution of ABO and Rh blood typing took place globally on the fringe of the standards. We note that 72.4% of the private laboratories and 48.9% of the public ones lacked at least one equipment and 51.3% at least one material for blood withdrawal; 38.2% of the laboratories did not respect blood withdrawal standards; 1.32% of the laboratories applied the 4×2 rule. The assessment revealed that respectively 10.8% and 30.7% of the blood centers and non-blood centers achieved the globular test solely; the same 40.5% and 46.2% used reagents of different brands. Anti-A1 and anti-H sera, and A1 and A2 red cells were not available in any laboratory. More than 64% of laboratories have senior technicians and biomedical analysis engineers but only 6.6% of the laboratories were directed by biologists, and 9.2% of the laboratories function with only one technician. ConclusionInstead of some assets, the laboratories assessment noted important non-conformities we ought to raise as a matter of urgency. It is a challenge whose resolution must give blood transfusion centers a reference position relatively to blood grouping when facing blood typing difficulties.

Preparation of a high titre Salmonella abortusovis H-antigen and antiserum and improvement of the microagglutination test

Serological diagnosis of small ruminant abortions due to Salmonella abortusovis (S.a.o) was improved by using modified procedures to produce high titre H-antigens and anti-H serum. While the titres of the two standard antigens were 1:8 and 1:20, both modified antigens had a titre of 1:50. Similarly, the use of adjuvanted and non adjuvanted antigens for the production of anti-H hyperimmune serum resulted in a titre of 1:160 000 as compared to 1:20 000 when standard procedures were used. In addition, the slow micro-agglutination test has been modified and results can now be read within the same day.

Isolation of fucosyl glycoproteins from human erythrocyte membranes by affinity chromatography using Aleuria aurantia lectin.

Fucosyl glycoproteins were fractionated from a sialoglycoprotein preparation of human erythrocyte membrane by using Aleuria aurantia lectin (AAL) coupled to Sepharose 4B. The affinity eluates were characterized as having high fucose content and significant H activity as measured in terms of N-acetylgalactosamine (GalNAc) incorporation with A1-enzyme and hemagglutination inhibition assay with anti-H sera, and the unadsorbed fractions contained low levels of fucose and were devoid of apparent H activity. Neuraminidase treatment of the material improved the recovery of the affinity eluate. Thus, 66% of the applied asialoglycoprotein was recovered in the eluate, though only 10% of the untreated material was bound and eluted. Moreover, a fucose-rich and H-active fraction was obtained through the affinity chromatography of the previously unbound fraction after neuraminidase treatment. In sodium dodecyl sulfate-gel electrophoresis, the main component of both the unadsorbed and eluted fraction was revealed to be PAS-1 glycoprotein. These results indicate that AAL-Sepharose was effective for isolating fucose-containing compounds from the membrane glycoprotein especially after neuraminidase treatment. The reasons for the appearance of H activity in the affinity eluates are discussed.

Purification, Morphology and Antigenic Characterization of Measles Virus Envelope Components

Measles virus haemagglutinin (H), fusion (F) and matrix (M) components were purified by affinity chromatography using monoclonal antibodies coupled to CNBr-activated Sepharose. H and M proteins were purified to homogeneity as determined by polyacrylamide gel electrophoresis by a single cycle of adsorption-desorption. The corresponding purification of the F protein required two cycles of adsorption-desorption. After the second adsorption an extra wash with 1 M-guanidine-HCl was employed to remove the contaminating cellular actin. Electron microscopic examination of the purified envelope proteins showed that, at pH 6.0, the H peplomers had a truncated conical shape (width 6.5 to 4 nm, length 16 nm), and the F peplomers had a club-like shape (dimensions of the oval head 6 X 9 nm, length 15 nm). Lengths of both peplomers were measured excluding their undiscernible hydrophobic part. The M component at pH 3.0 appeared as a rounded particle (diam. 8 nm, central accumulation of contrast 1.5 nm) suggested to include four to six M polypeptides. Rabbit hyperimmune sera were prepared against all three purified envelope components. These sera reacted only with the homologous antigen in radioimmunoprecipitation assays. Both antisera against the H and F components neutralized the virus and blocked virus-specific haemolysis, but only anti-H serum inhibited haemagglutination.

The secretor status of the foetus

One hundred amniotic fluids were tested for the presence of ABH blood group substances. Gestation at amniocentesis ranged from 12 to 28 weeks, with a mean of 16.6 weeks. The secretor status of the fluid was correlated with the secretor status of the baby after birth as determined from a sample of saliva. Fluid and saliva correlated in 98% of cases. It is felt that contamination with blood is likely to be the main source of error in typing amniotic fluids. However, because of the weaker activity of the anti-H serum, the detection of the presence or absence of H-substance can be doubtful if duplicate tests are not carried out. It is recommended that all fluids be checked for contamination with blood and that duplicate assays be done on two successive occasions. If these precautions are taken, the secretor typing of the amniotic fluid accurately reflects the secretor status of the individual foetus. Though probably not of wide application, the test could be of value in prenatal detection of dystrophia myotonica in individual cases.

Interaction entre allèles normaux A 1 ou A 2 et B chez l'hétérozygote AB : Étude comparative portant sur des familles européennes et africaines

A comparative study of European and African families is described to demonstrate interaction between A1 and A1B, A2 and A2B, B and A1B, B and A2B phenotypes who have either the same A allele or the same B allele. The quantitation of the agglutination has been determined by the "agglutination percentage measurement". The following reagents were used: human anti-A and anti-B, anti-A1 extracted from Dolichos biflorus and anti-H serum extracted from Ulex europaeus. In the A1 and A1B families--who received the same A1 allele--a weaker agglutination by anti-A1 and anti-A is observed in A1B Africans. In the A2 and A2B families who received the same A2 allele, the agglutination with anti-A is depressed from A2 to A2B in the European and African populations. In the B and A1B families who received the same B allele, a weakened agglutination between B and A1B with anti-B is only observed for the A1B Europeans. In the B and A2B families who received the same B allele, there is no variation of the agglutination between B and A2B with anti-B. The H status is the same for European and African populations, although the B Africans had less H but more B than Europeans. The results of our study led to the observation that the Africans have H deficient or a strong B gene. Like SSEBABI, we think that a strong B gene is perhaps acting more intensely on H substrate than A1.

Enzymic synthesis of a new type of fucose-containing glycolipid with fucosyltransferase of rat ascites hepatoma cell, ah 7974f

An α-fucosyltransferase activity has been demonstrated in rat ascites hepatoma AH 7974F cells catalyzing the transfer of l-fucose to asialo-GM 1 prepared from bovine brain GM 1 ganglioside to form a fucolipid in the presence of Triton X-100. The radioactive fucolipid was shown to be Fuc-(α 1→ 2)-Gal-(β1 → 3)-GalNAc-(β1→ 4)-Gal-(β1 → 4)-Glc-ceramide from the following results. The radioactive product coincided with authentic blood group H-active fucolipid from AH 7974F cell on thin-layer chromatography. The product formed a precipitation line not only with Ulex europeus lectin but also with eel anti-H serum on agarose gel plates. The terminal 14C-labeled fucose was released by Bacillus fulminans α (1→2)fucosidase as well as Charonia lampas α-fucosidase. The optimum pH value for the incorporation of l-fucose into asialo-GM 1 was 5.8 in sol|cacodylate/HCl buffer. The fucosyltransferase was highly specific for asialo-GM 1.

Studies on secretor specific H antigen of water-soluble blood group substances. I. Findings of anti-H(Se) precipitin in eel sera.

The presence of two kind of precipitins against blood group H substances from the human stomach and ovarian cyst was demonstrated in eel (Anguilla japonica) sera by immunodiffusion techniques in agar gel. One of them which forms the precipitation line to antigen side is the known anti-H precipitin containing anti-H agglutinin activity. The precipitating activity of the eel anti-H serum with H substance was inhibited by L-fucose and 2'fucosyllactose. The anti-H precipitin also reacts well with H substance from the hog stomach, but does not show clear precipitation lines with human secretor salivas and 1% solution of preparations of H substances from the small intestine, meconium and semen. Another precipitin which forms a precipitation line to the antiserum side in agar gel reacts well with H substances from human secretions and tissues. Because this precipitin reacted with water-soluble substances from human secretor, the antigen was named H(Se) and antibody anti-H(Se). The anti-H(Se) precipitin is not absorbed with group O red cells and the H and H(Se) activities of H substance against eel precipitins are decomposed by α-(1→2)-fucosidase.

Intestinal Colonization and Virulence of Salmonella in Mice

Within 3 h after oral challenge of mice with Salmonella typhimurium, foci of infection developed in the Peyer's patches of the small intestine. The numbers of organisms in the cecum, although in excess of those found in the small intestine, were not firmly associated with the cecal wall but were present largely in the cecum's contents. The Peyer's patches at first were remarkably incapable of eliminating even small numbers of Salmonella, but at about 7 days after infection developed the ability to eliminate a less virulent strain of S. typhimurium. Selected strains of Salmonella of varied virulence, and hybrid Escherichia coli/Salmonella typhimurium with varied O-antigens, revealed that those of low virulence could multiply within the intestinal Peyer's patches at nearly the same rate as a virulent strain, and the ability to multiply within the Peyer's patches was not dependent upon O-antigen type or smooth lipopolysaccharide. The ability of these strains to adhere to intestinal mucosa in vitro did not reflect on their ability to colonize the Peyer's patches, although strains of high in vitro adhesive ability appeared in greater numbers initially after oral challenge. Anti-O serum, ineffective in reducing the in vitro adhesive ability of virulent S. typhimurium, when given with the oral challenge prevented Peyer's patch colonization but was unable to prevent the appearance of a systemic infection. Anti-H serum, although effective in vitro in preventing adherence, had no effect in vivo. These experiments suggest that adhesiveness is neither essential nor sufficient for the virulence of Salmonella and that the usual development of a systemic infection after colonization of the small intestinal Peyer's patches may be subverted by the presence of O-antibody.

Isolation and purification of Lewis blood-group active glycosphingolipids from the plasma of human O Leb individuals.

Eight different glycosphingolipids with four to six sugars per molecule have been isolated from human plasma of blood-group O Leb secretores. Purfication was achieved by preparative highperformance thin-layer chromatography using four different solvent system, by gas-liquid chromatograhical analysis of the sugars as their alditol revealing integral numbers of sugars and by the hemagglutination inhibition test. Two blood-group Lea-active ceramide pentasaccharides (glycolipids 4,5) and three blood-Leb-active ceramide hexasaccharides (glycolipids 6, 7 and 8) were obtained, each revealing glucose, N-acetylglucosamine and fucose in molar rations of 1:2:1:1 (glycolipids 4,5) or 1:2:1:2 (glycolipids 6, 7 and 8) respectively. 0.027 μg of glycolipids 6, 7 and 8 completly inhibited the agglutination of human O Le(a-b+) erythrocytes by 50 μl of 4 hemagglutinating units of caprine anti-Leb sera. Honegeneity of glylipids 6, 7 and 8 was confirmed by the exclusion of contamination of the hypothetical Led antigen being a structural isomer of the Leb antigen: A crude neutral glycolipid fraction enriched in fucolipds from the plasma of O Le(a-b-) secretors showed a high-performance thin-layer chromatographical migration behaviour completely different from that of glycolipids 6, 7 and 8. Glycolipids 4 and 5 exhibited distinct Lea blood-group activities (0.4 μg of glycolipid 4 or 0.6 μ of glycopid 5 per 50 μl of 4 hemagglutinating units of caprine anti-Lea serum), but were slightly contaminated by blood-group H activity or blood-group Leb (7 μg of glycolipid 4/100 μl human anti-H serum and 1.7 μg of glycolipid 5/50 μl caprine anti-Leb serum). 0.01 μg, 0.0006 μg or even only 0.0001 μg of glycolipid 6, 7 or 8 respectively are sufficient for inclubation to convert 9x107 O Le(a-b-) erythrocytes into Lebpositive cells. This means in the case of glycolipid 8. that maximally 400 molecules per erythrocyle on the cell surface are sufficient to acheive an agglutinability by anti-Leb sera. In addition, two different globosides (glycolipids 1, 2) and a new ceramide tetrasaccharide (glycolipid 3) were isolated. The latter substance contains glucose, galactose and N-acetylglucosamine in molar ratios of 1:2:1, but differs from the lacto-N-neotetraosylceramide by showing a distictly slower high-performance thin-layer chromatographical migration behaviour.

Serotyping of Serratia marcescens: evaluation of Le Minor's H-immobilization test and description of three new flagellar H antigens

The H-immobilization test of Le Minor for determining flagellar (H) antigens was evaluated and compared with tube and slide H-agglutination tests. The test proved specific and easy to perform, and titration end points were clearly discernible. The degree of serological cross-reactivity between H antigen reference strains of Serratia marcescens was low. Consequently, this test was adopted for routine serological analysis of H antigens, using unabsorbed rabbit immune anti-H sera. As a result of using this procedure, three new provisional H antignes, designated H14, H15, and H16, are proposed.

A quantitative study of the ABH red cell antigens in the Kulamwo of Ivory Coast

The agglutination percentages given by various erythrocytes with reference sera have been compared in blood samples from Kulamwos living in the Ivory Coast and of blood donors of the regional transfusion centre of Toulouse. The study of O, A, B and AB phenotypes shows: 1. in O Kulamwos a higher agglutination percentage with anti-H than in Toulousains; 2. in B Kulamwos a higher agglutination percentage with anti-B than in Toulousains; 3. in A Kulamwos a very marked frequency of A intermediate which can be classified in different categories due to the variability of their agglutination percentage with anti-A1 and anti-H sera.

The H specificities of butanol extracts of human erythrocytes.

A previous report of other researchers that aqueous phase fractions of erythrocyte stromata of secretors contained more ABH blood group activity than similar fractions of non-secretors was confirmed. The recovery of H activity in the butanol phase extracts of both secretors and non-secretors, which had not been seen previously, is reported here for the first time. Three H specificities were present when the aqueous and butanol phases of group O secretor stromata were examined in inhibition of agglutination studies with anti-H of group Oh and group A1 sera and three lectins. The anti-H (group Oh) sera were inhibited by both aqueous and butanol fractions. Anti-H serum (group A1) and the lectin Ulex europaeus were inhibited by the aqueous fraction only. The lectins Laburnum alpinum and Lotus tetragonolobus were not inhibited by either the aqueous or butanol phase extracts.

α-Galactosyltransferase Activity in the Serum of Frogs (Rana catesbeiana)

Human blood group 0 cells were converted into B-active cells by incubation with uridine diphosphate D-galactose and unfractionated serum from bull frog (Rana catesbeiana).Under these conditions, «Bombay» (Oh) type red cells were not converted, and group 0 foetal cells possessing a few H antigenic sites were only weakly converted into B-active cells.In addition, the agglutination titer of the converted cells with anti-H serum decreased.These results therefore indicate that the conversion depends upon the presence of H-substance on the red cells.It was concluded from the results of this investigation that the blood group B specific α-galactosyltransferase was present in the serum of Rana catesbeiana as well as in the human group B serum.

The H Specificities of Butanol Extracts of Human Erythrocytes

A previous report of other researchers that aqueous phase fractions of erythrocyte stromata of secretors contained more ABH blood group activity than similar fractions of non-secretors was confirmed. The recovery of H activity in the butanol phase extracts of both secretors and non-secretors, which had not been seen previously, is reported here for the first time. Three H specificities were present when the aqueous and butanol phases of group O secretor stromata were examined in inhibition of agglutination studies with anti-H of group O(h) and group A(1) sera and three lectins. The anti-H (group O(h)) sera were inhibited by both aqueous and butanol fractions. Anti-H serum (group A(1)) and the lectin Ulex europaeus were inhibited by the aqueous fraction only. The lectins Laburnum alpinum and Lotus tetragonolobus were not inhibited by either the aqueous or butanol phase extracts.

Quantitative study of the ABO system in several groups of African populations.

Agglutination percentages of red blood cells from European and from three African populations (Twareg from Mali, Sara from Central African Republic, and Niokholonko from Senegal) in the ABO system have been compared. The results show a greater agglutinability of group O erythrocytes with anti-H sera, for African subjects than for Europeans. No significant difference was noted between African and European B subjects, except for the Niokholonko, whose agglutination percentage was slightly higher with anti-B. The study of group A disclosed a large number of Aint in all African populations studied. The AB African red cells seem to be less agglutinable with anti-A and anti-A1 sera than European ones; A and A1 antigens may be depressed by a strong B antigen.