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PubMed1999

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration.

We have developed a new helper adenovirus (Ad) based on serotype 2, Ad2LC8cCARP, for use in the Cre/loxP system (Parks et al. Proc Natl Acad Sci USA, 1996; 93: 13565-13570) to generate Ad vectors deleted of all protein coding sequences (helper-dependent Ad vectors (hdAd)). A comparison of Ad2LC8cCARP and our original helper virus (based on serotype 5, Ad5LC8cluc) showed that the two helper viruses amplified hdAd with a similar efficiency, and resulted in a similar yield and purity after large-scale preparation of vector. In vitro, the resulting hdAd2 had a similar transduction efficiency and expression kinetics of transgene (beta-gal) as the hdAd5. An important feature of the helper-dependent system is that all virion components, except the virion DNA, derive from the helper virus. Consequently, vectors produced with help from Ad2LC8cCARP were not neutralized by antibodies against Ad5, and vectors produced with Ad5 helper were resistant to neutralizing antibodies against Ad2. Analysis of transgene expression in mouse liver after intravenous injection of the Ad2-based hdAd showed that the vector could efficiently transduce the liver, and produce high levels of a foreign transgene, similar to those expressed by the hdAd generated with the Ad5 helper virus. Mice immunized with hdAd2 produced Ad2-neutralizing antibodies, which did not cross-react with hdAd5. To determine if successful repeat Ad vector administration could be achieved by sequential use of alternative Ad serotypes, we injected mice with hdAd2 (hSEAP) followed 3 months later by a lacZ-expressing hdAd of either the same or different serotype. Repeated administration of hdAd2 resulted in a 30- to 100-fold reduction in transgene expression compared with naive animals. In contrast, no decrease in transgene expression was observed when the second vector was of a different serotype. These results demonstrate that effective vector readministration can be achieved by the sequential use of hdAds based on alternative serotypes.

Adenoviridae↗
PubMed2006

High-performance liquid chromatography method for rapid assessment of viral particle number in crude adenoviral lysates of mixed serotype.

Accurate adenovirus (Ad) quantification requires labor- and time-intensive viral stock purification. While crude viral lysates can be titered by plaque assay, this cell-based assay is neither rapid nor accurate. Consequently, a method for quantification of crude, unpurified viral culture lysates is needed. Given growing interest in alternative Ad serotypes (different from well-studied and characterized serotype Ad5) for basic research and for therapeutic applications, such a method should also apply to alternative serotypes. Using a Q Sepharose XL (QSXL) column-based method, we describe a robust quantification method resulting in efficient retention of viral particles of all serotypes, while non-viral components of crude infected cultures remain largely in the flow-through. The high-performance liquid chromatography-QSXL method allows rapid, accurate adenoviral quantification in crude lysates as well as identification of the various serotypes present in mixed-serotype crude lysates. We also report on conditions that efficiently strip and regenerate the column, extending its functional life.

Adenoviridae↗
PubMed2000

16S to 23S rRNA spacer fragment length polymorphism of Salmonella enterica at subspecies and serotype levels.

The variation in the lengths of the internal transcribed spacer (ITS) between 16S and 23S rRNA genes of 101 strains representing 58 serotypes of Salmonella enterica (used for Salm. choleraesuis) subsp. enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV) and indica (VI) was used for typing by PCR amplification. Ten fragment lengths were observed by denaturing polyacrylamide gel electrophoresis on an automatic DNA sequencer resulting in 21 unique fragment patterns. Ten out of the 58 serotypes showed specific patterns but 48 serotypes were not fully differentiated. More than one ITS pattern was observed in seven serotypes. Five of the 21 fragment patterns contained representatives of more than one subspecies. Under non-denaturing electrophoresis conditions, serotype specificity was obtained but precise ITS fragment length determination was not possible. DNA sequence comparison between ITSs of individual rrn operons is needed to further interpret ITS diversity within Salm. enterica at serotype and subspecies levels.

Bacterial Typing Techniques↗
PubMed1997

Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri.

We have isolated the lysogenic bacteriophage SfII, which mediates glucosylation of Shigella flexneri O-antigen, resulting in expression of the type II antigen. SfII belongs to group A of the Bradley classification and has a genome size of 42.3kb. DNA sequencing of a 4 kb BamHI subclone identified four open reading frames (ORFs), of which only two were found to be necessary for serotype conversion. These genes were named bgt, which encodes a putative bactoprenol glucosyl transferase, and gtrII, encoding the putative type II antigen determining glucosyl transferase. These genes are adjacent to the integrase gene (int) and attachment site (attP), which are highly homologous to those of Salmonella bacteriophage P22. Another ORF encoded a highly hydrophobic protein of 120 amino acids with homologues in Escherichia coli, Salmonella bacteriophage P22 and S. flexneri. Previous studies identified gtrX, the glucosyl transferase gene, of bacteriophage SfX, which also glucosylates the O-antigen specifically. We determined that gtrX-mediated expression of the group 7,8 antigen also requires bgt. This allowed us to identify gtrII as being the serotype antigen II determining glucosyl transferase. Southern hybridization and polymerase chain reaction (PCR) analyses indicated that bgt homologues exist in the genomes of all S. flexneri serotypes and in E. coli K-12, whereas gtrII was only detected in strains of serotype 2. Transposon TnphoA-derived chromosomal mutations of bgt and gtrII in S. flexneri serotype 2a were isolated and characterized. [35S]-methionine labelling and the use of a T7 RNA polymerase expression system identified a protein of 34kDa corresponding to Bgt. However, GtrII, which has a predicted molecular weight of 55 kDa, was not detected. We propose that the function of Bgt is to transfer the glucose residues from the UDP-glucose onto bactoprenol and GtrII then transfers the glucose onto the O-antigen repeat unit at the rhamnose III position. The chromosomal organization of these serotype-converting genes, when compared with their homologues in E. coli K-12 chromosome and the P22 bacteriophage genome, were very similar. This suggests that the regions encode similar functions in these organisms and have a similar evolutionary origin.

Amino Acid Sequence↗
PubMed2001

Serotype and adhesion of Pseudomonas aeruginosa isolated from contact lens wearers.

The purpose of the present study was to correlate the serotypes of Pseudomonas aeruginosa to the bacterial adhesion to contact lenses and human corneal epithelial cells. Twenty-three strains isolated from contact lens wearers were used for the study. The bacterial serotypes were examined with a P. aeruginosa antisera kit. The attachment of bacteria on contact lenses or human corneal epithelial cells was determined by counting the number of adhered bacteria after incubation of the bacteria with contact lenses or corneal epithelial cells. The 23 ocular isolates belonged to seven serotypes. Strains of serotypes I, G and E were the three dominant serogroups and were more adhesive to contact lenses compared with other groups of the bacteria. The bacterial serotypes and the clinical sequelae were not strongly related. These results indicate that the surface characteristics of bacterial serotypes are related to the bacterial adhesion to the surface, but the pathogenesis of the bacteria may result from multiple factors.

Bacterial Adhesion↗
PubMed2000

Epidemiologic typing of Escherichia coli using RAPD analysis, ribotyping and serotyping.

OBJECTIVE: To compare random amplified polymorphic DNA (RAPD) analysis and ribotyping with serotyping for epidemiologic typing of Escherichia coli. METHODS: Thirty-two epidemiologically unrelated strains, nine cerebrospinal fluid isolates with the O7K1 serotype from nine patients, and nine sets of epidemiologically related E. coli isolates from nine patients were typed by RAPD analysis, ribotyping and serotyping. RESULTS: Among the 32 epidemiologically unrelated E. coli isolates, 29 types were distinguished by RAPD analysis, 25 by ribotyping and 27 by serotyping. Indistinguishable patterns were obtained by RAPD analysis and ribotyping within the collection of nine cerebrospinal fluid isolates. For the epidemiologically related isolates, intrapatient variation was only found by RAPD analysis among the isolates of one set and by ribotyping among the isolates of two sets. No interpatient variation was observed between three sets of isolates. With serotyping, the epidemiologically related isolates yielded similar typing relationships to those obtained by RAPD analysis and ribotyping. CONCLUSIONS: RAPD analysis had the highest discriminatory capacity for typing E. coli isolates. RAPD analysis, ribotyping and serotyping can all be used for assessment of strain relationships.

Adult↗
PubMed1998

An evaluation of ERIC PCR and AP PCR fingerprinting for discriminating Salmonella serotypes.

PCR fingerprints of 89 Salmonella isolates belonging to 22 serotypes were obtained using ERIC PCR (enterobacterial repetitive intergenic consensus PCR) and AP PCR (arbitrarily primed PCR) to evaluate the ability of different fingerprinting methods to differentiate or identify serotypes and subtypes. Fingerprints were scored and comparisons were made using a computer program. ERIC PCR produced a unique, complex fingerprint for almost every isolate, but these fingerprints did not identify serotypes. One AP PCR primer also produced complex fingerprints that discriminated among isolates, but again did not identify serotypes. A second AP PCR primer produced simple patterns, including one pattern shared by 35 isolates from 12 different serotypes. In general, the three sets of PCR fingerprints distinguished isolates, but were not correlated with serotypes. Matching fingerprints from different gels by computer was difficult, since similarities were based on both intense and faint bands. In addition, this study suggests that dendrograms created from PCR fingerprints should be viewed with caution.

Base Sequence↗
PubMed1999

The presence of muramidase released protein and extracellular factor protein in various serotypes of Streptococcus suis isolated from diseased and healthy pigs in Spain.

A total of 142 strains from different serotypes of Streptococcus suis isolated in Spain from diseased pigs (88 strains) and healthy carrier pigs (54 strains) were studied for the presence of a muramidase released protein (MRP) and an extracellular factor (EF). The following five phenotypes: MRP+EF+, MRP+EF-, MRP-EF+, MRP+EF* and MRP*EF- were detected. A high percentage of S. suis serotype 2 strains isolated from diseased pigs (84 per cent) belonged to phenotype MRP+EF+, but this phenotype has also been noticed in other serotypes (serotypes 1, 1/2 and 14). Both proteins were detected in S. suis serotype 2 strains (26%) isolated from healthy carrier pigs and one of both proteins in serotypes 1 and 14 (phenotype MRP+EF*). The isolation of S. suis strains from healthy pigs which have shown both proteins may support the epidemiological significance of these carriers in the maintenance, transmission and distribution of virulent strains within and between swine farms.

Animals↗
PubMed2006

Serotypes and analysis of distribution of Shiga toxin producing Escherichia coli from cattle and sheep in the lower North Island, New Zealand.

AIMS: To serotype a subset of Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep to determine whether any corresponding serotypes have been implicated in human diarrhoeal disease, both in New Zealand and worldwide, and to examine the distribution of STEC and enteropathogenic Escherichia coli (EPEC) amongst cattle (calves, heifers and dairy) and sheep (lambs, rams and ewes), to assess whether carriage of identified bacterial genotypes may be associated with a particular age of animal. METHODS: Recto-anal mucosal swabs (RAMS) were taken from 91 calves, 24 heifers and 72 dairy cattle, and 46 lambs, 50 ewes and 36 rams, from four sites in the Manawatu and Rangitikei regions of New Zealand. Strains of E. coli selected from primary isolation plates were subjected to a multiplex polymerase chain reaction (PCR), to determine the presence of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae). RESULTS: Overall, 186/319 (58.3%) animals sampled were positive for stx1, stx2, or eae isolates. More sheep (43.9%) were stx1-positive than cattle (2.7%; p = 0.036), and amongst sheep more lambs and ewes were stx1-positive than rams (p = 0.036). Amongst cattle, more calves and heifers were eae-positive than dairy cows (p = 0.030). Two or more different STEC were isolated from at least 28 (9%) animals (three cattle and 25 sheep), based on their stx/eae genotype. Enterohaemolysin genes were found in 39/51 (76%) isolates serotyped. Twenty-one different serotypes were detected, including O5:H-, O9:H51, O26:H11, O84:H-/H2 and O149:H8 from cattle, and O26:H11, O65:H-, O75:H8, O84:H-, O91:H-, O128:H2 and O174:H8 from sheep; O84:H-, O26:H11, O5:H-, O91:H- and O128:H2 serotypes have been associated with human disease. CONCLUSIONS: If nationally representative, this study confirms that cattle and sheep in New Zealand may be a major reservoir of STEC serotypes that have been recognised as causative agents of diarrhoeal disease in humans. Distribution of STEC and EPEC in cattle and sheep indicates that direct contact with, in particular, calves or their faeces, or exposure to environments cross contaminated with ruminant faeces, may represent an increased risk factor for human disease in New Zealand.

Adhesins, Bacterial↗
PubMed2000

Clonally related penicillin-nonsusceptible Streptococcus pneumoniae serotype 14 from cases of meningitis in Salvador, Brazil.

Active hospital-based surveillance in the city of Salvador, Brazil, from December 1995 through October 1998, identified 221 patients with confirmed pneumococcal meningitis. Of these 221 patients, 29 (13%) had isolates with intermediate-level resistance to penicillin. Infection with these penicillin-nonsusceptible isolates was significantly associated with age of <2 years (P<.0019), previous antibiotic use (P<.0006), and coresistance to trimethoprim-sulfamethoxazole (P<.0000). Serotype 14 was the most prevalent serotype (55.2%) of penicillin-nonsusceptible isolates. Strain typing by repetitive element BOX polymerase chain reaction (PCR) analysis showed that penicillin-nonsusceptible serotype 14 isolates had closely related BOX PCR patterns, whereas penicillin-susceptible serotype 14 isolates each had distinct, unrelated patterns. Penicillin-nonsusceptible serotype 14 isolates from Salvador and other Brazilian cities had similar BOX PCR patterns. These observations indicate that in Brazil a large proportion of cases of penicillin-nonsusceptible pneumococcal meningitis appear to be caused by a closely related group of serotype 14 strains that may have disseminated to widely separate geographic areas.

Adolescent↗
PubMed2000

Serum samples from infants vaccinated with a pneumococcal conjugate vaccine, PncT, protect mice against invasive infection caused by Streptococcus pneumoniae serotypes 6A and 6B.

Streptococcus pneumoniae serogroup 6 is an important cause of respiratory tract disease worldwide. Vaccination with 6B polysaccharide induces antibody response to the cross-reacting serotype 6A, but the protective capacity of 6A antibodies induced in infants remains unknown. In this study, passive immunization with serum samples obtained from infants vaccinated with an octavalent polysaccharide protein conjugate vaccine, PncT, protected mice against bacteremia and/or lung infection caused by intranasal challenge with serotypes 6B and 6A. Protective infant serum samples had significantly higher serotype-specific IgG levels and opsonic activity than did nonprotective serum samples. The protective level to either serotype was approximately 1 microg of specific IgG antibodies injected per mouse (corresponding to approximately 0.3 microg/mL). The protection was strongly related to opsonophagocytic antibody levels measured in vitro. These results demonstrate that PncT induces antibodies in infants that protect mice against invasive disease caused by the homologous serotype and by the cross-reacting serotype 6A.

Animals↗
PubMed2002

Antimicrobial use and serotype distribution of nasopharyngeal Streptococcus pneumoniae isolates recovered from Greek children younger than 2 years old.

The serotype distribution of 781 nasopharyngeal pneumococcal isolates recovered from 2448 unselected children aged 2-23 months was studied. Only 3.9% of the children for whom cultures were performed attended day care centers. The proportions of pneumococcal isolates that belonged to serotypes related to the 7-, 9- and 11-valent conjugate pneumococcal vaccine were 65%, 66%, and 70%, respectively. The pneumococcal carriage rate among untreated children was 34%; the rates among children treated with antibiotics during the periods 1-30 or 31-60 days before the time of nasopharyngeal sampling were 25% and 36%, respectively. There was a significant positive association between antimicrobial use and carriage of antibiotic-resistant pneumococci, which belonged mainly to vaccine-related serotypes. The proportion of isolates that belonged to vaccine-related serotypes in untreated carriers was 72%; however, the proportions in carriers treated 1-30 days or 31-60 days before sampling were 66% and 56%, respectively. In the nasopharynx, antimicrobial use selects for antibiotic-resistant pneumococci, mainly of vaccine-related serotypes, whereas it may promote an increase in the frequency of colonization with nonvaccine serotypes.

Anti-Bacterial Agents↗
PubMed2005

Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs.

The development of rotavirus vaccines that are based on heterotypic or serotype-specific immunity has prompted many countries to establish programs to assess the disease burden associated with rotavirus infection and the distribution of rotavirus strains. Strain surveillance helps to determine whether the most prevalent local strains are likely to be covered by the serotype antigens found in current vaccines. After introduction of a vaccine, this surveillance could detect which strains might not be covered by the vaccine. Almost 2 decades ago, studies demonstrated that 4 globally common rotavirus serotypes (G1-G4) represent >90% of the rotavirus strains in circulation. Subsequently, these 4 serotypes were used in the development of reassortant vaccines predicated on serotype-specific immunity. More recently, the application of reverse-transcription polymerase chain reaction genotyping, nucleotide sequencing, and antigenic characterization methods has confirmed the importance of the 4 globally common types, but a much greater strain diversity has also been identified (we now recognize strains with at least 42 P-G combinations). These studies also identified globally (G9) or regionally (G5, G8, and P2A[6]) common serotype antigens not covered by the reassortant vaccines that have undergone efficacy trials. The enormous diversity and capacity of human rotaviruses for change suggest that rotavirus vaccines must provide good heterotypic protection to be optimally effective.

Animals↗
PubMed2005

Increased prevalence of pediatric pneumococcal serotypes in elderly adults.

BACKGROUND: Pneumococcal disease is most prevalent among young children and elderly adults. We explored whether similarities exist in the serotypes that cause disease in these 2 high-risk groups. METHODS: With use of US population-based data from 1998-1999 (before the introduction of the 7-valent pneumococcal conjugate vaccine [PCV7] as routine immunization for infants) from the Centers for Disease Control and Prevention's Active Bacterial Core surveillance, we evaluated whether the rate of invasive pneumococcal disease caused by the pediatric serotypes (6B, 9V, 14, 19F, and 23F) increased among elderly persons. We adjusted for potential confounders in multivariable logistic regression. RESULTS: We analyzed 2987 pneumococcal isolates recovered from adults. The risk of infection with pediatric serotypes increased from 32.5% in 35-49-year-old persons to 51.2% in > or = 85-year-old persons (P < .001). Compared with 35-49-year-old persons, the risk of infection with pediatric serotypes was significantly elevated among 65-74-year-old persons (relative risk [RR], 1.68; 95% confidence interval [CI], 1.29-2.20) and increased progressively among persons aged 75-84 years (RR, 1.82; 95% CI, 1.41-2.36) and > or = 85 years (RR, 2.29; 95% CI, 1.72-3.05), with adjustment for sex, race, geographic location, underlying illness, and penicillin resistance. The rate of penicillin resistance was also elevated in the elderly population but was not significant after adjustment for serotype and race. CONCLUSIONS: The increased proportion of severe pneumococcal disease caused by pediatric serotypes in the elderly population might indicate opportunities for prevention with use of PCV7.

Adult↗
PubMed1999

Diagnosis of pneumococcal pneumonia in epidemiological studies: evaluation in Kenyan adults of a serotype-specific urine latex agglutination assay.

A serotype-specific tube latex agglutination assay for 10 pneumococcal serotypes was evaluated with use of urine samples from 72 Kenyan adults with pneumonia whose blood or lung aspirate cultures were positive for Streptococcus pneumoniae, 203 patients with pneumonia whose cultures were negative for S. pneumoniae, and 101 afebrile controls. Detection thresholds for purified capsular polysaccharide in normal urine ranged from 0.33 to 10 ng/mL. The sensitivity of the assay for the 10 pneumococcal serotypes was 0.57 (95% confidence interval, 0.44-0.70) and was unaffected by human immunodeficiency virus seropositivity, prior antibiotic use, and bacteremic or nonbacteremic status but varied significantly by serotype. Of the pneumococci obtained by culture, 81% were of serotypes (1, 4, 5, 6, 7, 9, 12, 14, 19, and 22) that were included in the antigen assay. Strong simultaneous agglutinations against two different serotypes were found in urine samples from two patients. The specificity of the assay was 0.98 (lower 95% confidence limit, 0.95). Subjective reading of agglutination results introduced variation in specificity that may be inapparent if not formally measured. The assay extended the diagnostic yield in pneumococcal pneumonia by a factor of 2.2 (from 54 diagnoses established by blood culture to 119 established by both methods) and may therefore prove useful in reducing the sample size of epidemiological studies of pneumococcal pneumonia in adults.

Adolescent↗
PubMed2003

Genetic relatedness, antibiotic susceptibility, and serotype distribution of Streptococcus pneumoniae responsible for meningitis in Poland, 1997-2001.

Streptococcus pneumoniae is the third most frequent etiologic agent of bacterial meningitis in Poland. Therefore, the objective of this study was to characterize S. pneumoniae isolates responsible for meningitis in Poland from 1997 to 2001 by determining their serotypes, antimicrobial susceptibility, and genetic relatedness. Among 36 serotypes and rough isolates, the most common were serotypes 3, 8, 19F, 6B, 4, and 23F, altogether accounting for 45.3% of cases. Penicillin-nonsusceptible pneumococci constituted 14.8% of all isolates. A high heterogeneity of pneumococcal isolates concerning serological features as well as genetic relatedness based on pulsed-field gel electrophoresis analysis was observed. With the exception of some serotypes (e.g., 3, 7F, 19F), isolates of the same serotype do not appear to be more closely related to each other than to isolates of different serotypes.

Adolescent↗
PubMed2005

Molecular characterization of Listeria monocytogenes of the serotype 4b complex (4b, 4d, 4e) from two turkey processing plants.

Most foodborne outbreaks of listeriosis have been found to involve a small number of closely related strains of Listeria monocytogenes serotype 4b. The ecology of these organisms and their reservoirs in nature or in the processing plant environment, however, remain poorly understood. Surveys of environmental samples from two turkey processing plants in the United States indicated presence of L. monocytogenes of the serotype 4b complex (serotype 4b and the closely related serotypes 4d and 4e). In addition, environmental and raw product samples from one plant repeatedly yielded isolates with genetic markers typical of two major serotype 4b epidemic clonal groups, ECI and ECII. The pulsed field gel electrophoresis (PFGE) profiles of these isolates, however, were clearly distinct from those of confirmed epidemic-associated strains. Furthermore, we observed minor but consistent differences in PFGE profiles of isolates that harbored ECI- or ECII-specific genetic markers, and that were obtained at different sampling times from the same plant. The findings suggest processing plant persistence (or repeated introductions) and genomic diversification of L. monocytogenes serotype 4b isolates that harbor ECI- or ECII-specific genetic markers. Such diversification would need to be taken into consideration in further efforts to elucidate the evolution and epidemiology of these organisms.

Animals↗
PubMed2004

Recombinant bivalent vaccine against foot-and-mouth disease virus serotype O/A infection in guinea pig.

In this study, two DNA fragments encoding amino acid (141-160)-(21-140)-(141-160) of the VP1 of FMDV (foot-and-mouth disease virus) serotype O and (138-160)-(21-40)-(138-160) of the serotype A FMDV were chemically synthesized. These two tandem-repeat fragments were ligated and transfected into prokaryotic expression vector pTrcHis A to construct pTH-O-A. The other vector called pTH-O-scIgG-A was constructed similarly only that the two tandem-repeat DNA fragments were linked by the bovine-IgG heavy chain coding sequence. Guinea pigs immunized with the two bivalent vaccines pTH-O-A and pTH-O-scIgG-A showed both specific antibody activity and T cell proliferation responses. FMDV challenge tests showed that 85% and 70% of guinea pigs vaccinated twice with 200 mg of the fusion protein of pTH-O-A were protected from FMDV serotype O and serotype A infection respectively. 70% and 57% of the guinea pigs immunized with the fusion protein of pTH-O-scIgG-A were protected from FMDV serotype O and serotype A infection respectively.

Animals↗