Ch. Lämmler

Genotyping of Staphylococcus aureus isolated from bovine mastitis

The present study was designed to comparatively investigate 25 Staphylococcus aureus strains isolated from bovine subclinical mastitis. The S. aureus strains, obtained from six different farms at five locations in one region of Germany, were characterized by phenotypic and genotypic methods. The S. aureus could be identified and further characterized by their cultural, biochemical and hemolytic properties. To analyze the epidemiological relationship the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNA, by PCR amplification of the gene encoding the 16S-23S rRNA intergenic spacer, by PCR amplification of the gene encoding the IgG binding region and the X region of protein A and by amplifying, and subsequent, digestion of the gene encoding staphylococcal coagulase. The macrorestriction analysis revealed five DNA restriction patterns with DNA patterns I, III and IV occurring in three, four, and three different farms, respectively. In addition, clones with different DNA patterns could be found within one herd. The PCR products for the spacer DNA, the spa gene encoding the X region of protein A and the coa gene encoding coagulase corresponded mostly to the pattern observed by DNA fingerprinting. Amplification of the gene encoding the IgG binding region revealed sizes of 620 bp for 20 of the isolates and 280 bp for four isolates indicating, for the latter, a deletion of segments in this region. These findings show, that single, widely distributed clones seemed to be responsible for cases of bovine subclinical mastitis found in one region of Germany.

Septicaemia in emerald monitors (Varanus prasinus Schlegel 1839) caused by Streptococcus agalactiae acquired from mice

The present study was performed to investigate both the identity and the source of the bacteria responsible for a fatal septicaemia observed in a group of three subadult emerald monitors (Varanus prasinus Schlegel 1839). The emerald monitors were necropsied and examined by light microscopy, including immunohistology, and by electron microscopy. Tissue samples were additionally submitted for bacteriological, virological and parasitological examinations. The virological and parasitological results were noncontributory, whereas the bacteriological investigation resulted in the isolation of gram-positive cocci which were characterized biochemically and serologically and by molecular analysis. The death of the emerald monitors was caused by a partially leukocyte-associated septicaemic infection with streptococci of serological group B of serotype V. Phenotypically and genotypically identical group B streptococci were isolated from the intestine of subadult mice, obtained from the feed used for the monitors. The genotypical characterization included an identical DNA fingerprint of strains of both origins, indicating the epidemiological relation between the feeding mice and the infections of the monitors.

Identification and characterization of Streptococcus agalactiae isolated from horses.

Seven group B streptococcal cultures isolated from three horses reacted with group B-specific antiserum, were CAMP positive, pigmented and showed the typical biochemical properties of Streptococcus agalactiae. The identification could be confirmed by PCR amplification of the 16S rRNA gene and a subsequent RsaI restriction pattern typical for S. agalactiae. In addition, the isolates were identified by amplification of species specific parts of the 16S rRNA gene, the 16S-23S rRNA intergenic spacer region and by amplification of the CAMP-factor (cfb) gene. Six isolates could be classified as serotype III/Rib, one isolate as serotype Ia/cbeta. The occurrence of the protein antigens Rib and cbeta could be confirmed by PCR amplification of the respective genes. The six isolates of serotype III/Rib were hyaluronidase negative, had a hylB gene with a size of 4.6 kb and an insertion element IS1548 of 0.98 kb. The isolate of serotype Ia/cbeta was hyaluronidase positive, had a hylB gene with a size of 3.3 kb and no insertion element IS1548. In addition, all seven isolates had the insertion element ISSag2 and the gene lmb encoding the laminin binding surface protein Lmb and the gene scpB encoding C5a peptidase. According to the present results the group B streptococci isolated from horses showed characteristics of human isolates of this species.

Distribution of capsular types 1 to 28 and further characteristics of Streptococcus suis isolates from various European countries

Most of the 150 Streptococcus suis isolates from pigs and ruminants used in this study grew under aerobic conditions and were alpha-hemolytic on sheep blood agar. Part of the cultures required an increased CO2 concentration. These cultures, representing the CO2- dependent ecovar of S. suis, were mainly beta-hemolytic on sheep and horse blood agar and gave a synergistic hemolytic reaction with staphylococcal beta-lysin. Similar to S. suis reference cultures, the routine isolates showed typical biochemical properties of this species. Few cultures, mostly those from ruminants, could be classified as the sorbitol and mannitol-positive ecovar. Formamide extracts of 81% of the cultures reacted with group D-specific antisera. Serotyping of the S. suis isolates revealed mainly capsular types 2, 1/2, 1, 5, 11, 13, 23, 3 and 15. The determination of antibiotic susceptibility revealed a high number of cultures to be resistant to clindamycin, erythromycin, kanamycin, neomycin, streptomycin and tetracycline. Cultural, biochemical and serological properties together with antibiotic resistance patterns could be used to characterize individual isolates of S. suis. This could be of importance in epidemiological studies.

Identification and Epidemiological Relationship of Rhodococcus equi Isolated from Cases of Lymphadenitis in Cattle

The present study was designed to comparatively investigate 10 Rhodococcus equi isolates from cases of lymphadenitis in cattle. The isolates could be identified by cultural and biochemical properties. By serotyping the R. equi isolates 9 and 1, cultures could be classified as Nakazawas serotypes 15 and 8, respectively. The isolates did not agglutinate rabbit erythrocytes, were uniformly susceptible to most of the antibiotics tested, did not contain plasmids nor expressed virulence-associated proteins and yielded identical patterns in protein fingerprinting. To further analyze the epidemiological relationships, the isolates were additionally subjected to DNA fingerprinting. This was performed by pulsed-field gel electrophoresis (PFGE) after digestion of the chromosomal DNA with the endonuclease AsnI. PFGE analysis of the chromosomal DNA revealed 4 DNA restriction groups with DNA pattern I with 7 isolates as predominant group and DNA pattern II to IV with one isolate, respectively. The present results indicate that a single R. equi clone belonging to Nakazawas serotype 15 and according to PFGE to DNA restriction pattern I of the present investigation seems to be responsible for most of the cases of lymphadenitis of cattle described in this study.

Typing of Staphylococcus aureus isolated from nasal carriers

Eighteen Staphylococcus aureus strains (9 pairs) were isolated from 9 apparently healthy persons (including 5 persons from medical staff) regarded as persistent nasal carriers in a 5-7-day or 3-5-month period, respectively. The 18 S. aureus strains were characterized phenotypically and by genotypic methods. Biochemical properties determined with a commercial identification system, production of haemolysins as well as most of the antibiotic resistance date revealed an identity between both strains of each pair. This putative identity was confirmed for most of the paired strains by phage typing and plasmid profiling for all 9 pairs by determination of the number of repeats in the X region of the spa gene and by macrorestriction analysis of the chromosomal DNA of the isolates. The present results indicate that a single clone of S. aureus might colonize continuously persistent nasal carriers over a 5-7 day period, even for a period of 3-5 months.

Pheno- and genotyping of Rhodococcus equi isolated from faeces of healthy horses and cattle.

The present study was designed to comparatively investigate 21 Rhodococcus equi isolates from the faeces of clinically healthy horses and cattle. The isolates were identified by cultural and biochemical properties and by PCR analysis. The latter, targeted to the gene coding for the 16S ribosomal RNA, revealed a species specific PCR product. The isolates were further characterised by serotyping with two typing systems, by haemagglutination tests and by plasmid and virulence protein profiling. Among the 21 cultures, four cultures contained plasmids, two of the four cultures expressed 15-17 kDa virulence proteins, no cultures contained 20 kDA virulence proteins. The 21 cultures were further analysed by DNA-fingerprinting. This was performed by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). The DNA-restriction patterns were different for most of the isolates indicating a clone heterogeneity among isolates from single farms. Serotyping, determination of virulence marker and PFGE analysis of R equi appeared to be useful for further characterisation of this species, possibly of importance for virulence estimation of single R equi isolates and for epidemiological studies.

Binding of Immunoglobulin G and Albumin to Streptococcus dysgalactiae

All 24 cultures of Streptococcus dysgalactiae investigated bound 125I-IgG, 13 cultures additionally interacted with 125I-albumin. Inhibition experiments with unlabelled IgG and albumin preparations from humans and various animal species indicated the specificity of the binding sites which showed characteristics of IgG Fc-receptors of type III and albumin-receptors of type c. IgG and albumin-binding proteins could be removed from the streptococcal surface by solubilization and subsequently isolated by affinity chromatography. The isolated binding proteins of S. dysgalactiae strains C 12 and C 8 obtained from IgG and albumin sepharose precipitated with IgG in immunodiffusion reactions, and in immunoelectrophoretic studies, and they reacted, after transfer onto nitrocellulose, with 125I-IgG or 125I-albumin and vice versa. Antisera produced against IgG-binding proteins of S. dysgalactiae C 12 inhibited binding of 125I-IgG and 125I-albumin. Solubilization of binding proteins by trypsinization yielded low molecular weight fragments with 125I-IgG but not with 125I-albumin binding activities. IgG-binding proteins isolated from S. dysgalactiae C 26 reacted with 125I-IgG but not with 125I-albumin, indicating the presence of 2 groups of type III Fc-receptors among S. dysgalactiae strains.

Distribution of the hyaluronate lyase encoding gene hylB and the insertion element is1548 in streptococci of serological group B isolated from animals and humans

The present study was performed to investigate streptococci of serological group B obtained from various sources and group B streptococcal reference strains for serotype, hyaluronate lyase enzyme activity, the occurrence of the hylB gene and the insertion sequence IS1548. All group B streptococci were identified by cultural, biochemical, and serological properties and by polymerase chain reaction amplification of species-specific parts of the 16S-23S rDNA intergenic spacer region, the 16S rRNA gene and the CAMP-factor (cfb) gene. Of the 73 group B streptococci investigated, 59 strains displayed hyaluronate lyase enzyme activity. All hyaluronate-lyase-positive strains and three phenotypically hyaluronate-lyase-negative strains had a hylB gene with an amplicon size of 3.3kb. Eleven of the 14 phenotypically hyaluronate-lyase-negative strains generated a hylB gene PCR product with a size of 4.6kb, and 10 of these strains displayed a IS1548 amplicon with a size of 0.98kb. The hyaluronate-lyase-negative isolates were mainly observed among group B streptococci of serotype III/Rib. All strains harbouring IS1548 had an additional copy of IS1548 located downstream of the C5a peptidase (scpB) gene.

Detection and interspecies-transformation of a β-Lactamase-encoding plasmid from Pasteurella haemolytica

Pasteurella haemolytica-cultures, isolated from cattle with respiratory diseases, were investigated for their biotype, serotype, antimicrobial resistance and plasmid content. A plasmid encoding a beta-lactamase could be demonstrated in 9 of 19 Pasteurella haemolytica-cultures. These 9 cultures, all belonging to biotype A and serotype 1, were resistant to ampicillin, carbenicillin, penicillin G and ticarcillin. The plasmid of the respective cultures proved to be identical upon Southern blot hybridization. It could be transformed into Escherichia coli 490 A where it expressed again a beta-lactamase-activity.