Biomedical subjects
R Higgins
Publications and source records attributed to R Higgins.
Distribution of Streptococcus suis capsular types in 1993.
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Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay.
A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was standardized for the detection of specific antibodies following vaccination with Streptococcus suis capsular type 2 bacterins. No statistically significant increase of antibody titers was detected in vaccinated piglets compared to the nonvaccinated control group, even if a minority of piglets demonstrated an important postvaccinal response. Three of four vaccinated sows showed a low antibody response to vaccine and specific immunity was detected in piglets of only one litter of these three sows. Passive protection studies showed that none of the sera from vaccinated piglets were protective for mice whereas serum obtained from hyperimmunized pigs gave protection.
Untreated milk as a source of verotoxigenic E coli O157.
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Detection of immunoglobulin-G-binding proteins in Streptococcus suis.
This study was undertaken to search for the presence of immunoglobulin G (IgG)-binding proteins in Streptococcus suis, an important swine pathogen. Whole bacterial cells were incubated with human or pig IgG conjugated to gold particles and examined by transmission electron microscopy. Cells of some S. suis strains were labelled as were cells of the positive control strain, Staphylococcus aureus Cowan I. Binding of pig and human IgG to five different bacterial species of group D streptococci, to reference strains representing the 29 capsular types of S. suis, and to 12 S. suis capsular type 2 strains was then examined using Western blotting. All strains interacted with pig and human IgG, although the binding profiles were slightly different. A 52 kDa protein was observed in all capsular types of S. suis. This protein, absent in other group D streptococcal species, was observed in all capsular type 2 isolates originating from diseased or clinically healthy pigs, and was shown to bind human IgG-Fc fragments. The IgG-binding activity was also observed in the culture supernatant and was sensitive to proteolysis.
Use of polyvalent coagglutination reagents for serotyping of Streptococcus suis.
Polyvalent coagglutination reagents (PRs) have been evaluated for the serotyping of Streptococcus suis. Monovalent antisera produced against 28 S. suis reference strains have been grouped to obtain five different pools. A total of 249 field isolates previously identified and belonging to different serotypes were tested with PRs prepared by two different procedures: (i) monovalent coagglutination reagents were individually prepared and mixed in equal proportions, and (ii) antisera were mixed in equal proportions before the addition of the Staphylococcus aureus suspension. Only antisera tested by a tube agglutination test with 2-mercaptoethanol and presenting titers of 1:32 or higher were used. Results obtained with PRs prepared by both procedures were similar, and there was a very good correlation between the capsular type of the isolate and the reaction obtained with PRs. Thus, from a practical viewpoint, it is suggested that PRs be prepared by the first procedure. To isolates, were tested in parallel with both the PRs and the monovalent coagglutination reagents over a 1-year period. Ninety-nine percent of the typeable and all of the untypeable isolates were correctly identified. Serotyping with PRs is suggested to be a very useful and reliable screening procedure, particularly when a large number of S. suis isolates have to be serotyped. In addition, the choice of antisera to be included in a given pool is facultative and should be oriented to the needs of a region or a country.
Septicemic Enterococcus infection in an adult llama.
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The effectiveness of gentamicin or polymyxin B for the control of bacterial growth in equine semen stored at 20 degrees C or 5 degrees C for up to forty-eight hours.
Semen from three stallions was used to evaluate the effectiveness of two antibiotics added to semen extender for samples stored at 20 degrees C or 5 degrees C for up to 48 hours. Each ejaculate was divided into six different treatments: semen+extender (SE); SE+gentamicin (100 micrograms/mL); SE+polymyxin B (1000 units/mL); and each of the above treatments inoculated with Pseudomonas aeruginosa ATCC 27853. Sampling of diluted semen for bacteriological analysis was performed after 2, 8, 24 and 48 hours of preservation at either temperatures. The presence of nonspecific bacteria was noted after two hours in all SE aliquots. The number of bacteria did not change in samples stored at 5 degrees C, while in samples preserved at 20 degrees C, it increased by three to four times after 48 hours. In semen aliquots treated with either of the antibiotics, the number of nonspecific bacteria was very low after two and eight hours at both temperatures. This number remained stable up to 48 hours at 5 degrees C, while an increase was noted at 24 and 48 hours at 20 degrees C. At 5 degrees C, the number of P. aeruginosa cells tended to decrease between 24 and 48 hours in SE aliquots. The presence of gentamicin or polymyxin B appeared to rapidly inhibit growth of P. aeruginosa. At 20 degrees C, growth of P. aeruginosa increased between 8 and 24 hours in SE, while the presence of antibiotics almost completely inhibited the growth of the bacterium.(ABSTRACT TRUNCATED AT 250 WORDS)
A sandwich enzyme-linked immunosorbent assay for the detection of Streptococcus suis.
A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and the identification of Streptococcus suis capsular types 1, 2, 1/2, 3 and 22. The specificity of this test was first evaluated using reference strains of S. suis capsular types 1 to 28 and 1/2 as well as 15 different bacterial species susceptible to be isolated from swine. The ELISA developed was very specific for capsular types 1, 3 and 22 but it could not discriminate between capsular types 2 and 1/2. In a second study, S. suis isolates from 328, 493, 368 and 76 diseased pigs were used to detect capsular types 1, 2 or 1/2, 3 and 22 respectively. The relative specificity and sensitivity varied between 98% and 100%. The ELISA results were in excellent agreement with the standard techniques (biochemical tests, coagglutination and capsular reaction tests) in detecting both positive and negative strains. Kappa values were 0.80, 0.99, 0.97 and 1.00 for detecting S. suis capsular types 1, 2 or 1/2, 3, and 22 respectively. To evaluate the relative-sensitivity of the test, primary cultures from 73 diseased pigs and tissue samples from 67 diseased pigs were used directly for detecting these capsular types. With primary cultures, the relative specificity and sensitivity (95.9% and 91.6% respectively) remained high and the test was very suitable (Kappa = 0.87). The ELISA using tissue samples gave a good specificity (97.6%), a moderate sensitivity (62.5%) and a low agreement with standard tests (Kappa = 0.64).(ABSTRACT TRUNCATED AT 250 WORDS)
Production of capsular material by Streptococcus suis serotype 2 under different growth conditions.
The procedure currently used for the production of Streptococcus suis antigen is very long and includes several subcultures. The aim of the present work was to study the in vitro production of capsular material by S. suis serotype 2 after each of these subcultures. The amount of capsular material produced was evaluated by electron microscopy using bacterial cells grown on blood-agar plates and in Todd-Hewitt broth (THB) or THB supplemented with serum. In addition, the production of antibodies in rabbits with antigens produced using different growth conditions was compared. Antigens produced after only three subcultures possessed as much capsular material as cells obtained after the complete procedure and induced a similar antibody response. The use of serum as a supplement to the broth did not assure a higher production of capsule; in addition, antibody titers obtained with antigens produced in THB were as high as those obtained with antigens produced in THB supplemented with serum. We recommend the use of three subcultures in nonsupplemented broth for the production of immunogens. This revised protocol offers two main advantages: it is less time-consuming because of the limited number of subcultures and is also less expensive since nonsupplemented broths are used.
Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates.
Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.
Production and characterization of two Streptococcus suis capsular type 2 mutants.
Two avirulent mutants of Streptococcus suis capsular type 2 (M2 and M42) were produced from a highly virulent strain. Mutant M2, obtained after serial subcultures of the parent strain in the presence of rabbit anti-capsular type 2 serum, no longer possessed the type-specific capsular antigen, as demonstrated by serotyping methods and immunoelectron microscopy. The Lancefield group D antigen could not be detected on the cell surface of this mutant using the immunogold labelling technique. SDS-PAGE of lysozyme treated cells demonstrated that a 44 kDa protein which was present in the parent strain, was absent in mutant M2. Immunoblotting using rabbit whole cell homologous anti-serum revealed that the protein was strongly immunogenic. Mutant M2 was totally avirulent in mice, and the homologous antiserum completely failed to protect mice against challenge with the parent strain. However, mutant M42, obtained after passages of the parent strain at 42 degrees C, remained capsulated but lacked the same 44 kDa protein as mutant M2. The quantity of sialic acid present in the capsule was similar to that of the parent strain. Despite the presence of antibodies against the capsule, antiserum prepared against M42 only partially protected mice against a challenge with the parent strain. The 44 kDa cell wall protein could act as a virulence factor as well as an important immunogen of S. suis capsular type 2.
Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec.
A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemagglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains of A. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 and were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.
A prospective, randomized, blind comparison of three biopsy techniques in the management of patients after renal transplantation.
Although conventional histology (CH) of needle core biopsies has been accepted as the gold standard for diagnosis of renal allograft rejection, this assumption has never been tested. Fine-needle aspiration cytology (FNAC) and monoclonal antibody (panleukocyte) staining of needle core biopsies (MABS) have been suggested to be superior to CH. A total of 50 patients received a cadaveric renal transplant followed by immunosuppression with triple therapy. Biopsies were taken routinely at days 7,14,21,28, and 90, with additional biopsies taken between these times if rejection was suspected (total biopsy sessions = 219). Specimens were taken for CH, FNAC, and MABS at each biopsy session, but only the result of one technique (previously randomly allocated) was communicated back to the clinical team, using a standardized grading system. Subsequently the presence or absence of rejection was determined by retrospective analysis of the clinical and biochemical course by 4 clinicians, without reference to the biopsy result. Graft survival was not significantly different in the three groups. The sensitivities for CH, FNAC, and MABS were 75%, 59%, and 77%, respectively, while the specificities were 87%, 96%, and 80%, respectively. Inadequate samples for analysis occurred frequently with the MABS technique--and, to a lesser extent, with CH--and both techniques tended to overdiagnose rejection. FNAC most often gave an answer but did miss clinically important rejection episodes. Needle-core biopsy processed for CH remains the most reliable biopsy technique for the diagnosis of rejection of renal allografts. FNAC is a useful technique for monitoring grafts with stable function or nonfunction. MABS does provide information equivalent to CH, but, in this study, had a high incidence of inadequate samples.
Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.
The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.
Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe.
This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.
Antimicrobial resistance patterns and plasmid profiles of Streptococcus suis isolates.
Streptococcus suis isolates recovered from diseased animals in Quebec and western Canada and from human cases in Europe were tested for their susceptibility to different antimicrobial agents and screened for their plasmid content. Most isolates from Quebec were clindamycin, erythromycin, and tetracycline resistant; animal isolates from western Canada were notably less resistant to clindamycin and erythromycin, whereas human isolates were considerably more susceptible to most antimicrobials tested. More than 60% of isolates had plasmids that ranged from 1.5 to 35 kilobases (kb). Of the 7 plasmid profiles found, 2 were particularly frequent in isolates from Quebec and western Canada, suggesting the presence of epidemic strains in the swine population. A particular plasmid band of about 5 kb was present in most Canadian isolates. When this band was used as a probe in colony and Southern blot hybridization, most isolates harboring the 5-kb plasmid hybridized, even though their plasmid profiles were different. Human isolates from Europe differed in their plasmid content from Canadian isolates of animal origin. Although a high degree of antimicrobial resistance was associated with the presence of plasmids in most isolates, it was not possible to establish a causative relationship.