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PubMed2004

PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II.

A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae.

Actinobacillus Infections↗
PubMed2001

Interpreting results from trials of pneumococcal conjugate vaccines: a statistical test for detecting vaccine-induced increases in carriage of nonvaccine serotypes.

Conjugate vaccines against Streptococcus pneumoniae (pneumococcus) protect against nasopharyngeal carriage of serotypes included in the vaccine. However, in several clinical trials, vaccinees have shown increased carriage of nonvaccine serotypes of pneumococcus. These increases may be due to serotype replacement, if vaccine-induced protection against carriage of vaccine serotypes increases susceptibility to carriage of nonvaccine serotypes. Alternatively, observed increases may be an artifact of "unmasking," in which nonvaccine serotypes are more readily detected among vaccinees than among controls because vaccine serotypes are not present. In this paper, a statistical test for distinguishing serotype replacement from unmasking is described. The test attempts to reject a null model of unmasking alone; serotype replacement is inferred if the observed increase in detectable nonvaccine serotype carriage among vaccinees is significantly greater than that expected under the null model. Significance is assessed using the Bayesian "posterior predictive p value" as modified by Robins et al. (J Am Stat Assoc 2000;95:1143-56). Analysis of data from a South African trial suggests that replacement may have occurred in the study, but results do not reach the conventional level of significance in rejecting the null hypothesis of unmasking (p = 0.074). The author performs sensitivity analyses for the prior and for unmeasured confounding by differences in susceptibility to pneumococcus carriage. The implications of the findings and the assumptions and limitations of this technique are then discussed.

Bayes Theorem↗
PubMed2006

A unique serine-rich repeat protein (Srr-2) and novel surface antigen (epsilon) associated with a virulent lineage of serotype III Streptococcus agalactiae.

Group B streptococci (GBS) are pathogens of both neonates and adults, with serotype III strains in particular being associated with invasive disease and meningitis. In this study, a novel GBS surface antigen, epsilon, was found to be co-expressed with the previously reported delta antigen on an identical subset of serotype III GBS. Expression of delta/epsilon on the surface of serotype III GBS was shown to distinguish the restriction digest pattern (RDP) III-3 and multilocus sequence typing (ST)-17 lineage. epsilon-Specific antibodies were reactive with a unique, high-molecular-mass, serine-rich repeat protein (Srr-2) found exclusively in RDP III-3 strains. The gene encoding Srr-2 was located within a putative accessory secretory locus that included secY2 and secA2 homologues and had a genetic organization similar to that of the secY2/A2 locus of staphylococci. In contrast, serotype III delta/epsilon-negative strains and strains representative of serotypes Ia, Ib, Ic and II shared a common Srr-encoding gene, srr-1, and an organization of the secY2/A2 locus similar to that of previously reported serotype Ic, delta/epsilon-negative serotype III and serotype V GBS strains. Representative serotype III delta/epsilon-positive strains had LD(90) values 3-4 logs less than those of serotype III delta/epsilon-negative strains in a neonatal mouse model of infection. These results indicate that the RDP III-3/ST-17 lineage expresses Srr-2 and is highly virulent in an in vivo model of neonatal sepsis.

Adenosine Triphosphatases↗
PubMed1984

Development of a Neisseria meningitidis group B serotype 2b protein vaccine and evaluation in a mouse model.

Although serotype 2 remains the predominant cause of group B Neisseria meningitidis disease in many parts of the world, most cases of this disease are now due to serotype 2b rather than 2a. For this reason, we adapted the serotype 2a vaccine method of C. E. Frasch and M. S. Peppler (Infect. Immun. 37:271-280, 1982) to the production of a serotype 2b protein vaccine. A spontaneously occurring nonencapsulated mutant of the group B serotype 2b strain 3006 was obtained by selection on group B antiserum agar. Serotype 2b outer membrane protein vaccines were prepared with less than 1% lipoplysaccharide contamination. The immunogenicity of these vaccines was evaluated in mice in the presence and absence of meningococcal group B and group C capsular polysaccharides. The group B and group C polysaccharides equally potentiated the antibody response to the serotype 2b protein. Addition of aluminum hydroxide or aluminum phosphate markedly improved the antibody response to the serotype 2b protein, but aluminum hydroxide-adjuvanted vaccines consistently elicited higher antibody levels. Aluminum hydroxide-adsorbed serotype 2a and 2b protein vaccines were evaluated for induction of cross-protective bactericidal antibodies. The 2a vaccines were 2a specific, whereas the 2b vaccines elicited antibodies strongly bactericidal for both 2a and 2b meningococcal strains and protected against bacteremia in a mouse model. It may therefore be possible to provide protection against both 2a and 2b disease by using an aluminum hydroxide-adsorbed protein vaccine containing a single serotype 2 protein component.

Adjuvants, Immunologic↗
PubMed1988

Virulence properties and protective efficacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5.

The role of the capsule of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 in bacterial virulence, and the protective efficacy of antibody to serotype 5 capsule was investigated. Encapsulated H. pleuropneumoniae serotype 5 were resistant to killing by complement and antibody to capsule or somatic antigens, whereas a noncapsulated mutant was sensitive to killing by the alternative complement pathway alone. Antiserum to whole H. pleuropneumoniae serotype 5 bacteria or monospecific antiserum to capsule was capable of opsonizing bacteria of the homologous serotype for phagocytosis by swine polymorphonuclear leukocytes but was not opsonic for a heterologous serotype. An immunoglobulin M monoclonal antibody to the serotype 5 capsule was not opsonic for any serotype. Mice were protected against lethal, intranasal challenge with the homologous or heterologous serotype after immunization with live encapsulated or noncapsulated bacteria, but not after immunization with killed bacteria, lipopolysaccharide, or a capsule-protein conjugate vaccine. The protection induced by immunization with live bacteria was transferred to nonimmune, syngeneic mice by serum but not by spleen cells. Nonimmune pigs passively immunized with monospecific swine serum to capsule were protected from lethal infection but not from development of hemorrhagic lung lesions, whereas pigs passively immunized with swine antiserum to live bacteria did not develop severe respiratory lesions. Thus, the capsule of H. pleuropneumoniae serotype 5 was inhibitory to the bactericidal activity of serum and was antiphagocytic. Antibody to the capsule was opsonic but was not fully protective.

Animals↗
PubMed1991

Serotype-specific epitope(s) present on the VP8 subunit of rotavirus VP4 protein.

cDNA clones representing the VP8 and VP5 subunits of VP4 of symptomatic human rotavirus strain KU (VP7 serotype 1 and VP4 serotype 1A) or DS-1 (VP7 serotype 2 and VP4 serotype 1B) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2 and VP4 serotype 2) were constructed and inserted into the pGEMEX-1 plasmid and expressed in Escherichia coli. Immunization of guinea pigs with the VP8 or VP5 protein of each strain induced antibodies that neutralized the rotavirus from which the VP4 subunits were derived. In a previous study (M. Gorziglia, G. Larralde, A.Z. Kapikian, and R. M. Chanock, Proc. Natl. Acad. Sci. USA 87:7155-7159, 1990), three distinct serotypes and one subtype of VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. The results obtained by cross-immunoprecipitation and by neutralization assay with antisera to the VP8- and VP5-expressed proteins suggest that the VP8 subunit of VP4 contains the major antigenic site(s) responsible for serotype-specific neutralization of rotavirus via VP4, whereas the VP5 subunit of VP4 is responsible for much of the cross-reactivity observed among strains that belong to different VP4 serotypes.

Animals↗
PubMed1981

Susceptibility of vaccinated swine and mice to generalized infection with specific serotypes of Erysipelothrix rhusiopathiae.

Swine were vaccinated with adsorbate bacterin made from Erysipelothrix rhusiopathiae of serotype 2 and were subsequently allotted to 4 exposure groups, each of which was exposed to 1 of the strains of E rhusiopathiae of serotypes 1, 2, 9, or 10. Mice were vaccinated with the same bacterin and were subsequently allotted to 60 exposure groups which were exposed to 60 strains of E rhusiopathiae, comprising 10 strains each of serotypes 1, 2, 4, 9, 10, and 11. Response to challenge of immunity in swine was determined by the presence of clinical signs of acute generalized erysipelas; response in mice was determined by the quantal (live-dead) method. Vaccinated swine were as susceptible to the strain of serotype 10 as were nonvaccinated control swine, whereas vaccinated swine were immune and control swine were susceptible to the strains of serotypes 1 and 2. The strain of serotype 9 was not sufficiently virulent to induce acute generalized erysipelas, even in control swine. Arthritis was not prevented by vaccination, but its frequency and severity were less in vaccinated swine exposed to strains of serotype 1 or 2 than in those exposed to strains of serotype 9 or 10. Vaccinated mice were significantly (P less than 0.05) more susceptible to the strains of serotype 10 than to those of any other serotype tested.

Animals↗
PubMed1993

Purification of a Pasteurella haemolytica serotype 1-specific polysaccharide epitope by use of monoclonal antibody immunoaffinity.

A murine IgM monoclonal antibody causing bacterial agglutination was used in an immunoaffinity procedure to purify a serotype 1-specific polysaccharide epitope from Pasteurella haemolytica. The P haemolytica serotype 1-specific antibody was precipitated from peritoneal ascitic fluid, dialyzed, and covalently attached to cyanogen bromide-activated Sepharose 4B beads. Retention of purified antibody activity and coupling efficiency were > 99% when evaluated by ELISA, agglutination testing, and protein determination. Potassium thiocyanate was selected as an eluant on the basis of reversible dissociation of bacterial agglutination and was titrated for the lowest effective concentration. Immunobead activity was observed microscopically by immobilization of encapsulated P haemolytica serotype 1 and its reversible dissociation after elution with 0.4M potassium thiocyanate. Specificity of immobilization was visualized, using P haemolytica serotypes 2 and 5, which were not bound, and by blocking serotype-1 binding with homologous capsular material. Saline-extractable capsular material from P haemolytica serotype 1 was used as an antigen source. After elution of the serotype 1-specific polysaccharide epitope, the product was dialyzed and analyzed, using chemical and immunologic methods. The immunoaffinity product contained no detectable protein and greater than half the original hexosamine content. Using defined monoclonal antibodies in ELISA, titration of the original capsular material and the immunoaffinity product revealed specific retention of lipopolysaccharide, a 10- to 30-kd polysaccharide antigen common to all P haemolytica and P multocida serotypes, and serotype 1-specific capsular polysaccharide, indicating possible epitope sharing among polysaccharide antigens of P haemolytica serotype 1.

Agglutination↗
PubMed2001

Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma.

CONTEXT: Human papillomavirus (HPV) infection has been established as a cause of cervical cancer. Epidemiologic studies suggest that Chlamydia trachomatis infection also confers increased risk for cervical squamous cell carcinoma (SCC). Whether this risk is serotype-specific is unknown. OBJECTIVE: To study the association between exposure to different C trachomatis serotypes and subsequent development of cervical SCC. DESIGN AND SETTING: Longitudinal, nested case-control study within a cohort of 530 000 women who provided samples to serum banks in Finland, Norway, and Sweden. The data files were linked to respective national cancer registries. SUBJECTS: One hundred twenty-eight women who had developed invasive cervical SCC at least 12 months following serum donation. Each case had 3 matched controls. MAIN OUTCOME MEASURE: Risk for the development of cervical SCC by IgG antibodies to 10 different C trachomatis serotypes, adjusted for antibodies to HPV types 16, 18, and 33 and for serum cotinine levels. RESULTS: Of specific C trachomatis serotypes, serotype G was most strongly associated with SCC (adjusted odds ratio [OR], 6.6; 95% confidence interval [CI], 1. 6-27.0). Other serotypes associated with SCC were I (OR, 3.8; 95% CI, 1.3-11.0) and D (OR, 2.7; 95% CI, 1.3-5.6). Presence of serum IgG antibodies to more than 1 serotype increased the adjusted ORs for SCC (P<.001 for trend). CONCLUSIONS: Chlamydia trachomatis serotype G is most strongly associated with subsequent development of cervical SCC. Increasing numbers of exposures to different C trachomatis serotypes also increases risk. Our results strengthen the evidence that there is a link between past C trachomatis infection and cervical SCC.

Antibodies, Bacterial↗
PubMed1994

Mapping of antigenic sites involved in serotype-cross-reactive neutralization on group A rotavirus outercapsid glycoprotein VP7.

Two neutralizing monoclonal antibodies (N-mAbs) were utilized to locate amino acid (aa) residues involved in the formation of serotype-cross-reactive epitopes on the VP7 of selected group A rotaviruses. N-mAb 954/159/13 neutralized G serotype 3 as well as porcine G serotype 4 rotaviruses, whereas N-mAb 57/8 neutralized G serotype 3, 4, 6, 9, and 10 strains. Neutralization-resistant variants of each serotype were selected in the presence of these two monoclonal antibodies. Sequence analysis of the gene encoding VP7 of such variants revealed: (i) variable regions VR-5 (aa 88-100), VR-8 (aa 209-223), and VR-9 (aa 235-242) are involved in cross-reactive neutralization; (ii) an aa substitution can occur at the same position on the VP7 of different serotypes selected by a given N-mAb; (iii) the location of an aa substitution on the variant VP7 selected by a given N-mAb can vary depending on the rotavirus serotype; (iv) a substituted single aa species at a specific position on the variant VP7 selected by a single N-mAb can vary, resulting in variants which exhibit antigenic differences; and (v) the VP7 of a porcine rotavirus Gottfried strain has a unique antigenic mosaic of serotype 3 and serotype 4.

Amino Acid Sequence↗
PubMed1995

Rotavirus serotypes and electropherotypes in Finland from 1986 to 1990.

Four epidemic seasons of rotaviruses were studied in Helsinki during 1986-1990. This is the first Scandinavian study, where both electropherotypes and serotypes are determined. Out of 5316 fecal specimens 769 (14.5%) rotavirus positive samples were detected by electron microscopy. Of these, 645 isolates (83.9%) gave a clear RNA pattern in gel electrophoresis and they clustered into 87 electropherotypes. An illustrative number of isolates representing each electropherotype (= E-type) was serotyped using VP7 protein-specific monoclonal antibodies for serotypes G1-G4 and without exceptions, within one E-type only a single serotype specificity was found. After establishment of the serotype of each E-type, the distribution of serotypes was scored as 61.2%, 2.0%, 0.5% and 29.8% for G1-G4, respectively; 6.5% remained untypable. Two seasons had one predominant E-type (Season 1, 1986-87, and Season 3, 1988-89, 84.2% and 80.6% of rotavirus positive samples, respectively). Both were followed by a season with no predominant E-type, but several minor E-types. Altogether, 5 short E-types (13/645 samples) with serotype G2 specificity were found, most of them occurring in Season 2. Only 2 E-types (3 samples) belonged to serotype G3. Group C rotavirus was found in 8 specimens. In this study a shift in serotypes, from G1 to G4, was observed in Finland in 1988/89; a similar shift was reported in many European countries at that time.

Child↗
PubMed1982

Recovery of dual serotypes of bluetongue virus from infected sheep and cattle.

Dual serotypes of bluetongue virus (BTV) were recovered from field-collected samples of sheep and cattle blood. Two sheep, each infected with both BTV serotypes 10 and 17, were found in a flock with bluetongue disease associated with these two serotypes. One sheep infected with BTV serotypes 11 and 17 was found in a second flock; it was the only viremic sheep detected and was clinically ill. Dual serotype infections of one beef and two dairy cattle were found in three geographically separate herds: mixtures recovered were of BTV serotypes 10 and 17 and serotypes 11 and 17. Clinical signs of illness were absent in the cattle in two herds, but severe conjunctivitis was seen in several cows in a third herd, including the cow with a dual serotype infection (BTV 11 and 17). Two of the cattle with dual infections had no serological evidence of BTV as determined by the agar gel precipitin test; serum was not available from the other cow with a dual serotype infection. The significance of dual infections and immune tolerance are discussed.

Animals↗
PubMed2005

Serotyping of hepatitis C virus in hemodialysis patients: comparison with a standardized genotyping assay.

The aims of this study were to investigate the prevalence of hepatitis C virus (HCV) genotypes and serotypes in anti-HCV-positive hemodialysis patients and determine the concordance between genotyping and serotyping methods. Sixty-two hemodialysis patients were included in this study. HCV RNA was determined using polymerase chain reaction assay and genotypes using a line probe assay. HCV serotyping was performed with competitive enzyme-linked immunosorbent assay. Genotype 4 (52 patients) was the most predominant genotype, followed by type 1 (10 patients). The most prevalent HCV serotype was type 4 (41 patients), followed by serotype 1 (6 patients). We detected multiple serotypes in 4 patients and untypeable strains in 11. The overall sensitivity of serotyping assay was 82% for the study patients. According to the genotyping results, the sensitivity of serotyping was 60% and 86.5% for HCV types 1 and 4, respectively. There was a 100% concordance between results of serotyping and genotyping in the identification of HCV type 1 and 91% concordance in HCV type 4. Serological typing method may be of great value in microbiology laboratories that require a simple assay for identification of HCV genotypes, although the sensitivity of this assay may be limited by the immunocompetence of infected hemodialysis patients.

Adult↗
PubMed2006

Determination and geographic distribution of Orientia tsutsugamushi serotypes in Thailand by nested polymerase chain reaction.

Clotted blood samples of 240 scrub typhus patients were collected from 8 Regional Medical Sciences Centers in Thailand during 1999 to 2002. The serotypes of Orientia tsutsugamushi and their geographic distribution were determined. A nested polymerase chain reaction (PCR) was used to identify the serotypes of O. tsutsugamushi. The number of patients with positive results for O. tsutsugamushi was 25.0%. Two serotypes, Karp and Kato, were detected in these samples. No Gilliam serotype was detected from any of the study locations. The PCR products were sequenced using an automated DNA sequencer. The nucleotide sequence of gene encoding 56-kDa protein from these samples showed a high sequence homology with the reference sequence of O. tsutsugamushi Karp and Kato serotypes. O. tsutsugamushi Karp serotype was predominant throughout Thailand with the percentage of 96.8% of the total serotype-positive patients, whereas 3.2% for Kato serotype was observed only in the south. The highest number among the region of Karp serotype-positive patients of 31.6% was found in the northeast.

Adolescent↗
PubMed2006

Serotype-specific detection of adeno-associated virus during laboratory preparation.

Adeno-associated virus (AAV) is a small single-stranded DNA member of the family Parvoviradae with at least eight recognized human serotypes, several of which are being studied as candidate vectors for gene therapy. When multiple serotypes are handled in the same laboratory, it is critical to know the serotype of a sample with certainty. Here, a rapid and reliable PCR-based method is presented for the identification of serotypes 2, 3B or 6 and for screening for cross contamination. The PCR assay is based on the use of differentially annealing, serotype-specific primer pairs targeted to the hypervariable regions of the capsid coding region of the AAV genome. Identity is determined by the presence of a PCR product of size specific to each serotype. Multiplexing the reaction allows all serotypes to be queried in a single reaction tube and greatly diminishes the presence of false positives. The method is capable of detecting as little as 25 fg of a contaminating serotype and is potentially extensible to other AAV serotypes.

Base Sequence↗
PubMed2000

Validation of a reverse transcriptase multiplex PCR test for the serotype determination of U.S. isolates of bluetongue virus.

Bluetongue (BT) is an arthropod-borne viral disease affecting ruminants primarily in tropical and temperate regions of the world. Of the 24 serotypes of BT virus (BTV) identified worldwide, five have been found in the United States. Serotype identification of BTV isolates is important to the epidemiology of the virus, but current methods are cumbersome. A single-tube multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) assay, previously developed for the serotype determination of U.S. BTV isolates, was evaluated. The determination of serotype was based on the size of the resultant amplified product. The procedure was evaluated using all 24 serotypes of BTV and nine serotypes of epizootic hemorrhagic disease virus (EHDV), a closely related orbivirus. Only the five U.S. serotypes of BTV were detected by the mRT-PCR. The assay was further tested using 132 BTV isolates originating from 24 western and southern states of the United States, from several different host species, spanning a period of 24 years. The serotypes of the isolates were determined by both a virus neutralization (VN) procedure and the mRT-PCR. Comparison of the mRT-PCR to the standard VN showed that the mRT-PCR successfully identified the serotypes of 130 of the isolates and was shown to be more reliable and specific than the VN assay.

Animals↗
PubMed2003

Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds.

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of antibodies against Actinobacillus pleuropneumoniae (Ap) serotypes 2, 6 and 12. The assay was designated MIX-ELISA. Lipopolysaccharide (LPS) from Ap serotypes 2, 6 and 12 was purified using hot phenol-water extraction followed by fractionation by size-exclusion chromatography. A mixture of fractions containing molecules with molecular weight above 50 kDa from all three serotypes was used as antigen. The MIX-ELISA was evaluated with sera from pigs experimentally infected with the serotypes 1, 2, 5b, 6, 7, 8, 10 and 12 of Ap biotype 1. In addition to reaction with sera from pigs inoculated with Ap serotypes 2, 6 and 12, reaction was observed with sera from pigs inoculated with serotype 8. Furthermore, the sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6 or 12 and with sera from herds free of infection with any Ap serotype of biotype 1. The ELISA showed a high herd sensitivity (0.98; 95% confidence interval: 0.89-1.00) and specificity (0.95; 0.88-0.99). The high diagnostic sensitivity and specificity of the assay indicate that screening of herds for Ap infection can be performed using this ELISA. Efficient serological surveillance can be achieved by using such mixed antigen ELISAs coated with size-selected LPS-antigens from the most prevalent serotypes.

Actinobacillus Infections↗
PubMed1997

Prevalence of capsular serotypes among Staphylococcus aureus isolates from cows with mastitis in the United States.

Development of an appropriate Staphylococcus aureus vaccine for bovine mastitis has eluded researchers for decades. The ability of S. aureus to form a protective exopolysaccharide capsule has posed a major obstacle because of the multiple serotypes and the poor immune response elicited by exopolysaccharides. This study characterized S. aureus serotypes isolated from cases of bovine mastitis obtained from veterinary diagnostic laboratories that service 44% of the dairy cattle in the United States. Major milk producing areas of the northeast, north central, Pacific coast and southwest were proportionately represented. Sub-samples of mastitic milk that contained S. aureus were frozen and sent to our laboratory for strain serotyping. The only other regional serotyping of S. aureus from bovine mastitis to date was done in France. The primary serotypes found were types 5 (51%) and 8 (18%) and 31% were non-typeable. In the current study, serotype 5 accounted for 18% of the isolates and serotype 8 for 23%. More importantly 59% of the isolates were not typeable with either type 5 or 8 antisera. These data indicate that S. aureus vaccines employing serotypes 5 and 8 would only be marginally effective in the United States. These data also suggest that development of a S. aureus vaccine for bovine mastitis should take into account regional variation in S. aureus serotypes.

Animals↗