Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope.

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.

Broad-range bacteriophage resistance in Streptococcus thermophilus by insertional mutagenesis.

Streptococcus thermophilus is a lactic acid bacterium used in industrial milk fermentation. To obtain phage-resistant starters, S. thermophilus strain Sfi1 was submitted to mutagenesis with the thermolabile insertional vector pG(+)host9:ISS1 followed by a challenge with the lytic S. thermophilus phage Sfi19. Vector insertions into four distinct sites led to a phage-resistance phenotype. Three mutants were characterized further. They were protected against the homologous challenging phage and 14 heterologous phages. All three mutants adsorbed phages. No intracellular phage DNA synthesis was observed in mutants R7 and R71, while mutant R24 showed a delayed and diminished phage DNA synthesis compared to the parental Sfi1 strain. In mutant R7 a short deletion occurred next to the insertion site which removed the upstream sequences and the 15 initial codons from orf 394, encoding a likely transmembrane protein. Analogy with other phage systems suggests an involvement of this protein in the phage DNA injection process. In mutant R24 the vector was inserted into orf 269 predicting an oxido-reductase. When the vector sequence was removed via homologous recombination across the duplicated insertion elements, mutant R24 returned to the phage susceptibility of the parental strain. This observation suggested that inactivation of orf 269 was not crucial for the resistance phenotype. A gene encoding a likely restriction subunit of a type I restriction-modification system was located directly downstream of the insertion site in mutant R24. hsdM and hsdS genes encoding the modification and specificity subunits of a type I R-M system and biological evidence for an active R-M system were detected in strain Sfi1, suggesting involvement of a type I R-M system in the resistance phenotype of R24.

Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory.

A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.

Effect of malnutrition on serum and milk antibodies in Zairian women.

Serum and human milk antimicrobial antibody titers were measured longitudinally in 17 malnourished and 14 control Zairian women during 6 to 18 months of lactation to test whether malnutrition is specifically associated with an impaired secretory antibody response. No decreases in total serum and human milk immunoglobulin concentrations, neutralizing antibody titers against rotavirus, or specific enzyme-linked immunosorbent assay antibody titers against rotavirus, respiratory syncytial virus, Escherichia coli, Streptococcus pneumoniae, and Haemophilus influenzae were detected when malnourished women were compared with control women. Malnutrition had no effect on circulating and secretory antibody concentrations in Zairian women. Daily human milk outputs, however, were about 30% lower in malnourished than in control women, resulting in a correspondingly lower ingestion of immunoglobulins by the children of malnourished women.

Effect of age on concentrations of serum antibodies to viral, bacterial, and food antigens in elderly Swiss people.

Serum antibody concentrations to two viral, five bacterial, and two food antigens were investigated in 307 elderly Swiss subjects, and the hypothesis of whether serum antibody titers decreased with age was tested. The cross-sectional part of the study consisted of 216 unselected consecutive patients hospitalized in one geriatric hospital. The patients were divided into two age groups (65 to 84 and 85 to 102 years old), and their antibody titers were compared. No age-related decreases in antibody titers were observed. The members of the two age groups were well matched for medical diagnosis and nutritional and inflammatory status. The prospective part of the study consisted of 91 healthy elderly subjects living in the community; they were 71 to 76 years old when they were enrolled in the study. Their serum antibody status was measured at the beginning of the study and 4 years later. We observed a significant decrease in diphtheria antitoxin levels and a significant increase in antibody titer to the capsular polysaccharide of Streptococcus pneumoniae. No change in antibody titer to rotavirus, respiratory syncytial virus, lipopolysaccharide of Escherichia coli, C polysaccharide of S. pneumoniae, or the polyribosyl-ribitol phosphate of Haemophilus influenzae was observed. Thus, no signs of B-cell immunosenescence were seen in these two groups of elderly Swiss people.

Effect of malnutrition in Ecuadorian children on titers of serum antibodies to various microbial antigens.

The titers of serum antibodies to natural infection with enteric and respiratory pathogens, to a food antigen and to tetanus and diphtheria toxoid were evaluated by enzyme-linked immunosorbent assay in 1,554 Ecuadorian children younger than 5 years of age. The nutritional status of the children was assessed by anthropometry and measurement of biochemical status indicators. The children were enrolled in a representative national nutrition and health survey. Antibody titers were analyzed as a function of the nutritional status of the children. For 12 of 14 antibody concentrations tested, underweight children showed lower antibody titers than did control children. The difference was statistically significant for antibody to both T-cell-dependent antigens (tetanus toxoid, rotavirus, respiratory syncytial virus) and T-cell-independent antigens (lipopolysaccharide, polyribosyl-ribitol phosphate, capsular polysaccharide). When children with a recent episode of diarrhea were excluded, many of the differences remained significant. When these children were further classified by age, only difference in titers of antibodies to respiratory syncytial virus and tetanus toxoid remained significant. No statistically significant difference was detected between underweight and control children with respect to protective antibody levels to four bacterial antigens. Anemic children showed significantly lower antibody levels to both T-cell-dependent and T-cell-independent antigens than did control children, and a higher proportion of anemic children had diphtheria antitoxin below a conservatively defined protective antibody level. No major differences in antibody titers were seen between children with different retinol and zinc concentrations in serum.

Detection and classification of Streptococcus thermophilus bacteriophages isolated from industrial milk fermentation.

In the last 30 years, 81 Streptococcus thermophilus bacteriophage isolates were collected from industrial yogurt (n = 40) and cheese (n = 41) fermentation. Forty-six distinct restriction patterns of phage DNA (11 in yogurt and 35 in cheese) were observed. The phages were investigated for host range, serological properties, and DNA homology to study whether these three independent techniques can be used to classify the phages into taxonomic groups. Yogurt factory-derived phages were classified into the same two subgroups by serology, host range analysis, and hybridization with subgroup-specific DNA sequences. Cheese factory-derived phages, however, could not be classified: the 35 cheese phage isolates with distinct restriction patterns showed 34 different host ranges. All but one cheese phage isolate showed serological cross-reactivity with yogurt phages. A phage DNA fragment that hybridized with all phage DNA samples was cloned, establishing the genetic relatedness of all S. thermophilus phages from our collection. With the sequence information from an unusually conserved S. thermophilus phage DNA element (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994), a PCR-based phage detection method was developed for cheese whey from a factory that produced mozzarella cheese with complex undefined starter mixes. PCR allowed the detection of phages in cheese whey (detection limit, 10(3) PFU/ml) which could not be detected by dot blot hybridization techniques (detection limit, 10(7) PFU/ml).

Bovine rotavirus V1005 a P5, not a P12, type like all viruses in a German survey.

Bovine rotavirus (BRV) V1005, like 34 further cell culture-adapted strains in a 6-year survey in Upper Bavaria, Germany, is not a P12 but a P5 P-type rotavirus. The conclusion is based on dot blot hybridization with P1-, P5-, and P11-specific cDNAs, encompassing the VP8* region of major sequence diversity, and on PCR using P1-, P5-, and P11-specific primer pairs derived from the VP5* region of VP4 (VP5* and VP8*, respectively, are the larger and smaller tryptic cleavage products of VP4). Sequencing of the hyperdivergent region of VP4 confirmed the close relatedness of BRV V1005 to BRV UK, the P5 prototype virus.

Coproantibodies to rotavirus serotype 1 infection in German children.

One hundred and ninety-eight serial stool samples were collected from 27 infants and children hospitalized in Bochum, Germany with gastroenteritis due to serotype 1 rotavirus (RV). RV antigen and RV-specific antibodies (Ab) (IgA ELISA and RV Wa-neutralizing Ab) were measured. The prevalence of RV-Ab positive stool samples in RV patients did not differ from that in stool samples from 80 control patients (40% and 42% for ELISA Ab and 11% and 6% for neutralizing Ab, respectively). None of the patients was breastfed in the week preceding stool collection. No significant increase in the prevalence of RV-Ab was observed between stool samples obtained during the early and late phase of hospitalization. We observed patients that continued to excrete RV antigen in the presence of neutralizing stool activities, and patients that showed cessation of RV stool excretion and resolution of clinical symptoms in the absence of RV-neutralizing activity in the stool samples.

Distinct Streptococcus thermophilus bacteriophages share an extremely conserved DNA fragment.

A cross-hybridizing 2.2-kb EcoRI fragment was cloned from two lytic Streptococcus thermophilus bacteriophages with distinct phenotypes. The DNA fragments, which contained two unidentified open reading frames, differed at only 3 of 2207 nucleotide positions. Partial sequencing of a temperate S. thermophilus phage and of a further lytic phage belonging to a different lytic group isolated 20 years earlier from a different geographical area confirmed this extreme sequence conservation. Hybridization of this phage DNA with bacterial host DNA was not observed. The evolutionary implications of these observations are briefly discussed.

Antibody response to polyribosyl-ribitol phosphate antigen of Haemophilus influenzae in Ecuadorian and German children.

Serum samples from 1,221 Ecuadorian children 0 to 5 years of age and from 236 German subjects were tested by enzyme-linked immunosorbent assay for class-specific antibodies to the capsular polysaccharide of Haemophilus influenzae type b (PRP antigen). A gradual prevalence increase of and mean titer increase in immunoglobulin M (IgM) antibody was seen in Ecuadorian but not in German children older than 6 months. At the end of the first year of life, about 50% of the Ecuadorian children showed IgM and IgG antibody to PRP. Seroepidemiological analysis revealed that living at a low altitude and lower calorie intake (a proxy measure of breast-feeding) were factors associated with earlier acquisition of PRP antibody. Children from low-altitude areas of Ecuador also experienced significantly more episodes of significant respiratory infections. The acquisition of PRP-reactive antibodies in Ecuadorian children might thus reflect exposure to encapsulated H. influenzae type b in lower respiratory tract infections.

Tetanus and diphtheria immunization coverage in Ecuadorian children after a national vaccination campaign.

Between October 1985 and June 1986, a national vaccination campaign was launched in Ecuador. Between March and November 1986, 7798 Ecuadorian children < 5 years old were enrolled in a representative health survey. According to their vaccination cards, 65% of children had a complete series of three doses of diphtheria-tetanus-pertussis (DTP) vaccination. Serum samples were obtained from a subset of 1400 children; by ELISA, 80% had serologic evidence of recent DTP vaccination and > 80% of the newborns had tetanus antitoxin titers, indicating recent tetanus vaccination of their mothers. For all 1400 children, median tetanus and diphtheria antitoxin titers were 1.0 and 0.6 IU/mL, respectively. These data indicate the possibility of success of such vaccination campaigns if supported by the government.

Rotavirus-inhibitory activity in serial milk samples from Mexican women and rotavirus infections in their children during their first year of life.

A total of 75 children born in rural Mexico were followed for diarrheal diseases and rotavirus (RV) excretion during the first year of life. For 18 children, an average of 14 serial breast milk samples were obtained between days 2 and 360 after delivery and were tested for RV-inhibitory activity. Of these samples, 70, 62, and 85% showed inhibitory activity against serotype (ST) 1 human RV, ST4 human RV, and ST3 simian RV, respectively; the median titers were 10, 10, and 20, respectively. Some 89% of the milk samples showed RV-specific antibodies in an enzyme-linked immunosorbent assay (median titer, 20). Surprisingly, 98% of the milk samples inhibited ST6 bovine RV. ST6, but not ST1, RV-inhibitory activity survived heat treatment (10 min at 80 degrees C). Of the 18 children tested, 13 children experienced 23 episodes of diarrhea (enterotoxigenic [n = 8] and enteropathogenic [n = 3] Escherichia coli, Campylobacter jejuni [n = 4], Shigella flexneri [n = 2], RV [n = 1]) and 5 children experienced 6 RV infections. Only one RV infection was associated with diarrhea. The five RV excretors did not differ from the nonexcretors with respect to the RV-inhibitory activity in the breast milk fed to them. The RV-inhibitory titers were too low in the majority of the studied Mexican milk samples to indicate an important effect of breast-feeding on the take rate of oral human, simian, or reassortant RV vaccines. Breast-feeding might, however, inhibit the take rate of a bovine RV vaccine.

Seroprevalence of immunoglobulin M (IgM) and IgG antibodies to polysaccharides of Streptococcus pneumoniae in different age groups of Ecuadorian and German children.

The age-specific prevalence of serum immunoglobulin M (IgM) antibody to capsular polysaccharides of Streptococcus pneumoniae, as detected by enzyme-linked immunosorbent assay, was studied in 1,301 Ecuadorian children enrolled in a national nutrition and health survey. This prevalence was 6% in infants < 6 months old and increased to 28% in children 6 to 11 months old, 49% in those 12 to 17 months old, and 58% in those 18 to 23 months old. About 80% of the 5-year-old children had this antibody. When tested separately against six different capsular polysaccharides, serum IgM antibody reacted with decreasing frequency with serotype 3, 8, 19, 6, 23, and 1 capsular polysaccharides. We did not observe a broadening of the antibody response with increasing age in the sense that more and more serotypes were recognized. A similar age-related prevalence was found for IgM antibody to the species-specific C-polysaccharide of S. pneumoniae and for IgG antibody to capsular polysaccharides of S. pneumoniae. A smaller German serum collection showed a comparable age-related prevalence of pneumococcus-specific serum IgG and IgM antibodies. The highest incidence of respiratory diseases was observed in 1- and 2-year-old Ecuadorian children. It thus seems that acquisition of serum antibody to S. pneumoniae reflects more the developmental maturation of an immune response than an actual exposure to different pneumococcal serotypes.

Reactivity of human serum antibody with lipopolysaccharide O 78 antigen from enterotoxigenic Escherichia coli.

Fifteen and five of 20 volunteers challenged with the enterotoxigenic Escherichia coli strain O 78.H11 showed a fourfold titre increase of serum ELISA antibody to the homologous O 78 and the heterologous O 8 lipopolysaccharide antigen, respectively. Sixty-three of 191 sera from 1- to 48-month-old German children showed serum antibody reactive with O 78 antigen, all but two of these O 78-positive sera also showed reactivity with at least one further O antigen. Only 14 of the O 78 reactive sera also showed antibody to heat-labile enterotoxin. In addition, soluble O 8 antigen could inhibit the binding of serum antibody to absorbed O 78 in 68% of the German children. Antibody reactive with O 78 antigen is thus not a reliable serological marker for enterotoxigenic E. coli infection in German children.

Neutralizing serum antibodies to serotype 6 human rotaviruses PA151 and PA169 in Ecuadorian and German children.

Serum samples from 726 Ecuadorian children who underwent natural rotavirus (RV) exposure were tested for neutralizing serum antibodies against two serotype 6 (ST6) human RV (HRV) isolates from Italy, PA151 and PA169, and two ST6 bovine RV (BRV) isolates, NCDV and UK. Gene 4 was distinct in all four ST6 strains. Ninety-one, 56, 67, and 65 serum samples neutralized HRV PA151 (13%), HRV PA169 (8%), BRV NCDV (9%), and BRV UK (9%), respectively. A total of 44 of the 91 serum samples which neutralized HRV PA151 did not neutralize the other three ST6 RV strains. In addition, we identified three serum samples that neutralized HRV PA151 but none of the six human or four animal RV STs. However, we failed to identify serum samples that neutralized HRV PA169 without neutralizing at least one of the major HRV STs. With a hospital-based serum collection from German children (excluding gastroenteritis patients), we identified 3 out of 197 serum samples tested that neutralized HRV PA151 specifically, whereas none neutralized HRV PA169 exclusively. None of the 71 German infants hospitalized with primary RV gastroenteritis showed a PA151- or a PA169-specific antibody response.

Antibodies to seven rotavirus serotypes in cord sera, maternal sera, and colostrum of German women.

Forty percent of colostrum samples from German women showed neutralizing antibody titers of greater than or equal to 50 to rotavirus (RV) serotypes 1, 3, 4, and 6. Antibody to serotypes 2, 8, and 9 was less prevalent. Titers are, however, too low to indicate an important effect of colostrum on the RV vaccine take rate. On the other hand, about 50% of the cord serum samples showed high neutralizing-antibody titers to serotypes 1, 3, and 4, which could interfere with the take rate of RV vaccines based on these serotypes in very young infants.

Infectious gastroenteritis does not act as a triggering mechanism for the synthesis of serum IgG antibody to beta-lactoglobulin.

beta-Lactoglobulin (BLG)-specific serum IgG antibody was measured by enzyme-linked immunosorbent assay in 1,392 serum samples from newborn to 5-year-old Ecuadorian children enrolled into a representative nutrition and health survey. At a 1:100 serum dilution, 62% of the children showed specific antibody (blank-corrected optical density greater than or equal to 0.1). This prevalence did not change with increasing age. More specifically, we did not observe a prevalence or titer increase of BLG-specific antibody in age groups where the majority of these Ecuadorian children experienced infection with rotavirus (8-24-month age groups) and enterotoxigenic Escherichia coli (8-12-month age group). In addition, BLG-specific antibody did not differ between children who did or did not experience an episode of diarrhea 15 days before blood sampling. We observed a small but statistically significant difference in BLG-specific antibody between subsamples of Ecuadorian children regularly or only occasionally ingesting milk. Titers were higher in the group consuming more milk.

Cattle develop neutralizing antibodies to rotavirus serotypes which could not be isolated from faeces of symptomatic calves.

Neutralizing antibodies against 10 serotypes of rotavirus were measured in sera from different age groups of German cattle. Only five of 143 sera did not neutralize heterologous serotypes. Sera from 64 of 76 calves younger than 1 year neutralized bovine rotavirus NCDV (serotype 6). From these calves, sera 54, 26, 51, 24, 12, 10 and 37, in neutralized addition, the heterologous serotypes 1, 2, 3, 4, 5, 7 and 9, respectively. Thirty-eight of 46 rotavirus isolates from Bavarian calves with diarrhoea were serotyped by neutralization: 22, 2 and 14 isolates were typed as serotype 6, serotype 10 (B223) and a newly defined subtype of serotype 10 (V1005), respectively. All serotype 6 isolates and none of the serotype 10 or V1005-like viruses tested hybridized to a NCDV-specific cDNA probe. Eight isolates gave equivocal results by neutralization. We failed however to identify serotype 1, 2, 3, 4 or 8 bovine rotavirus isolates by neutralization with hyperimmune sera and dot blot hybridization with serotype-specific cDNA probes. Thus cross-reacting antibodies in cattle might not represent an anamnestic response, but the recognition of a cross-reacting neutralization epitope shared by many rotavirus serotypes.

Neutralizing antibodies to heterologous animal rotavirus serotypes 5, 6, 7, and 10 in sera from Ecuadorian children.

Serum samples from 870 Ecuadorian children who underwent natural rotavirus exposure were tested for neutralizing serum antibody to heterologous animal rotavirus (RV) serotypes. Six percent of the sera neutralized porcine RV OSU (serotype 5), 10% neutralized bovine RV NCDV (serotype 6), 4% neutralized avian RV Ch-2 (serotype 7), and 8% neutralized bovine RV V1005 (serotype 10). Neutralization was defined as a 90% reduction in infectious virus at a 1:100 serum dilution. The prevalence of antibody to all four heterotypic viruses increased with the age of the children and the number of human RV serotypes neutralized, but prevalences did not differ significantly between children from rural and urban areas of Ecuador. No serum sample that specifically neutralized bovine RV NCDV was identified. We inferred from the seroepidemiological analysis that human RVs contain immunorecessive neutralization epitopes that can stimulate cross-neutralizing antibody to heterotypic animal RVs. This occurs increasingly with age and with the number of human serotypes recognized by a child's neutralizing antibody. Thus, it appears that a broadened immune response to the heterotypic strains occurs with repetitive RV infections.