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The present study gives a detailed phenotypic and genotypic characterization of three Trueperella abortisuis strains isolated from a ten year old male Hovawart dog with an abscess of anal sac, from urine of an eight year old European shorthair cat with urolithiasis and nephrolithiasis and from a 14year old Maine Coon cat with a perianal abscess, respectively. All three strains could be identified phenotypically, by MALDI-TOF MS analysis and genotypically by sequencing the 16S rDNA and the molecular target genes gap and tuf. The present study gives a first description of T. abortisuis of this origin.

In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals.

In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be observed with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in laboratories with less specified equipment.

In the present study a Trueperella (Arcanobacterium) bernardiae strain isolated from an anal swab of a three-day-old piglet could be identified phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing the 16S rDNA, the 16S-23S rDNA intergenic spacer region (ISR) and by sequencing the superoxide dismutase A encoding gene sodA. The present study gives the first information about the presence of T. (A.) bernardiae in specimen of animals.

The present study was designed to identify phenotypically and genotypically 61 Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. The A. pyogenes isolates showed the typical cultural and biochemical properties of this species and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the superoxide dismutase A encoding gene sodA of reference strains representing 8 species of genus Arcanobacterium and subsequent design of A. pyogenes sodA gene-specific oligonucleotide primer. The A. pyogenes sodA gene-specific oligonucleotide primer allowed, together with previously described A. pyogenes 16S-23S rDNA intergenic spacer region-specific oligonucleotide primer, a reliable molecular identification of all 61 A. pyogenes of various origins. The additionally performed PCR-mediated amplification of 5 known and putative virulence factor encoding genes revealed that 100, 20, 87, 75, and 98% of the A. pyogenes carried the genes plo, cbpA, nanH, nanP, and fimA, which allowed an individual strain characterization. This might help to elucidate the role the putative virulence factors play in bovine mastitis and in various other infections caused by this bacterial pathogen.

The aim of the present study was to characterize genotypically 45 Staphylococcus aureus strains isolated from humans, bovine subclinical mastitis and food samples in Argentina by rep-PCR and PCR amplification of virulence genes. Resistances to various antibiotics could be observed for the human S. aureus, less pronounced for the bovine strains, but not for the eight S. aureus isolated from food samples. The strains could be classified genotypically by rep-PCR and by amplification of the genes encoding protein A, coagulase, clumping factor, the collagen adhesin domains A and B, capsular polysaccharide 5 and 8, the accessory gene regulator agr classes I to III, and the S. aureus gene regulator sae. rep-PCR analyses and the different gene patterns revealed that the strains could be divided into seven groups mostly matching with the origin of the isolates. The present study describes genotypic variations of S. aureus strains isolated from different origins in Argentina. The study provides a valuable insight into molecular specificities of this important pathogen.

The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, O., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., Lammler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147-152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector.

Methicillin/oxacillin resistance of 10 S. intermedius strains was investigated by conventional and molecular methods. The strains tested had been isolated in Germany during routine veterinary microbiological examinations of specimens from a small animal clinic between May and September 2005. Epidemiological relationships of the strains were studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). Species identity of the 10 S. intermedius strains was confirmed by conventional methods and by PCR mediated amplification of S. intermedius specific segments of thermonuclease encoding gene nuc. As controls, four methicillin/oxacillin resistant S. intermedius (MRSI) strains obtained from specimens sent by four veterinarians and three selected methicillin/oxacillin sensitive S. intermedius (MSSI), also obtained from the small animal clinic, were tested. The 10 strains, representing approximately 6% of all S. intermedius isolated from the clinic throughout the time period mentioned above, and the four MRSI obtained from veterinarians, were methicillin/oxacillin and penicillin resistant using disk diffusion tests and could be cultivated on oxacillin resistant screening agar base (ORSAB). Both resistances could be confirmed by multiplex PCR detecting the resistance genes mecA and blaZ. The three MSSI were methicillin/oxacillin sensitive in all tests. Epidemiological investigation by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 10 MRSI strains obtained from the clinic and the four MRSI strains obtained from veterinarians, in contrast to the three MSSI strains, represent identical or closely related bacterial clones possibly indicating a cross-infection of the animals in the clinic and the distribution of a single MRSI clone in the pet population.

A marine bacterium associated with soft coral Sinularia polydactyla collected from Bandengan water, Jepara, North Java Sea, Indonesia, was successfully screened for antibacterial activity against pathogenic bacterium Streptococcus equi subsp. zooepidemicus K6.72 isolated from infected monkey of the island of Bali and identified based on morphological, biochemical and molecular methods. Marine bacterium was identified as Pseudomonas sp. based on its 16S rDNA and was found to amplify gene fragments of Non-ribosomal peptide synthetase (NRPS). Cloning and subsequent sequencing, a 360 bp long DNA fragment was obtained and the deduced amino acid sequence showed conserved signature regions for peptide synthetases and revealed a high similarity of 61.1% to genes peptide synthetase of Bacillus subtilis

In the present study 28 Arcanobacterium pluranimalium strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene pla encoding pluranimaliumlysin. No comparable reaction could be observed with control strains representing five species of genus Arcanobacterium and five species of genus Trueperella. The presented pla LAMP assay might allow a reliable and low cost identification of this bacterial pathogen also in laboratories with less specified equipment.

The present study was designed to comparatively investigate 19 Staphylococcus aureus strains isolated from specimens of 19 different birds during routine microbiological diagnostics. The S. aureus strains were characterized genotypically by polymerase chain reaction (PCR) amplification using 62 different oligonucleotide primers amplifying genes encoding staphylococcal cell surface proteins, exoproteins and two classes of the accessory gene regulator agr. All 19 investigated S. aureus were positive for the gene segment encoding a S. aureus-specific part of the 23S rRNA, the genes encoding thermostable nuclease (nuc), clumping factor (clfA) and coagulase (coa) and the gene segments encoding the Xr-repetitive region and the immunoglobulin G (IgG)-binding region of protein A (spa). In addition, all tested strains were positive for the genes hla and fnbA and negative for the genes seb, sec, sed, see, sej, tst, eta and etb. The remaining genes, including sbi, hlb, fnbB, ebpS, cna (domains A and B), cap5, cap8, set1, agr class I, agr class II, sea, seg, seh and sei were detected in a variable number of isolates. The presented data give an overview on the distribution of virulence determinants of S. aureus strains isolated from birds. This might be useful to understand the role of these virulence determinants in bird infections.

Summary The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA–seeI multiplex polymerase chain reaction.

Species-specific PCR tests, based on the manganese-dependent superoxide dismutase A encoding gene (sodA) and the chaperonin 60 encoding gene (cpn60), were developed for the identification of Streptococcus phocae, a bacterial pathogen of seals. The selection of both oligonucleotide primer pairs was performed after amplification and sequencing of internal parts of both genes using universal oligonucleotide primers. The sequence studies of both genes additionally confirmed that S. phocae could taxonomically be classified to the pyogenic group of the genus Streptococcus.

Bacteriological investigations of seals of the German North and Baltic seas resulted in the isolation of bacteria of the genus Streptococcus belonging to Lancefield's serological groups C, F, and L. According to biochemical, serological, and 16S ribosomal DNA analysis, the group C and group F streptococci were identified as Streptococcus phocae . The group L streptococci could be classified as Streptococcus dysgalactiae subsp. dysgalactiae .

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.

Streptococcus dysgalactiae serogroup C, G and L strains were investigated by polymerase chain reaction (PCR) using oligonucleotide primers designed according to species-specific parts of the 16S-23S rDNA intergenic spacer region. The oligonucleotide primers with specificity for the 16S-23S rDNA intergenic spacer region allowed a correct identification of all S. dysgalactiae serogroups C, G and L strains investigated. No cross-reactivities could be observed with any of the control strains indicating the usefulness of PCR-technology to identify the serologically heterogeneous species S. dysgalactiae.

Summary The 16S–23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae , S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis , S. phocae , S. uberis , S. parauberis , S. pyogenes and S. equi subsp. equi , respectively. The amplicons of S. equi subsp. zooepidemicus , S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae , S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis , S. phocae , S. pyogenes and S. suis , 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi , S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA Ala ), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus , S. porcinus and S.suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.

Streptococcus uberis and Streptococcus parauberis reference strains and isolates obtained from routine diagnostics were investigated by PCR with oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene, the 23S rRNA gene, and the 16S-23S rRNA intergenic spacer region of both species. All three primer pairs allowed an identification of 67 isolates as S. uberis and 4 isolates as S. parauberis .

An Arcanobacterium haemolyticum strain isolated from a postcastrational lesion of a horse was identified phenotypically and genotypically. The latter was performed by sequencing the 16S-23S rDNA intergenic spacer region (ISR), by amplification of the gene encoding A. haemolyticum phospholipase D, by amplification of A. haemolyticum specific parts of ISR-23S rDNA and by amplification of the newly described CAMP factor family protein encoding gene of A. haemolyticum. This indicates (as described previously for seven additional A. haemolyticum strains; Hassan et al. 2009) that A. haemolyticum seems to occur also in infections of horses.

The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 S. equi subsp. zooepidemicus and two S. equi subsp. equi could be amplified by PCR and sequenced. The sequence of the V2 region of eight S. equi subsp. zooepidemicus strains appeared to be identical or almost identical to the sequence of the two S. equi subsp. equi strains. The sequence of the remaining three S equi subsp. zooepidemicus strains differed significantly from the sequence of S. equi subsp. equi. These differences allowed a division of S. equi subsp. zooepidemicus strains into two 16S rRNA types and might possibly have consequences for the taxonomic position of these phenotypically indistinguishable strains of one subspecies. A molecular typing could additionally be performed by amplification of the gene encoding the 16S-23S rRNA spacer region. A single amplicon of the spacer gene of 1100 bp could be observed for one S. equi subsp. zooepidemicus, an amplicon of 950 bp for two S. equi subsp. equi strains and 10 S. equi subsp. zooepidemicus strains, a amplicon of 780 bp for 27 S. equi subsp. zooepidemicus strains and a single amplicon of 600 bp for one S. equi subsp. zooepidemicus strain. The variations of the V2 region of the 16S rRNA gene and the size variations of the 16S-23S rRNA spacer gene were not related to each other. Both variations could be used for molecular typing of this species, possibly useful in epidemiological aspects.

A total of 57 bacteria representing eight species of genus Arcanobacterium (A.) were investigated for hemolytic properties on blood agar containing sheep and rabbit blood and for CAMP-like reactions. An enhanced hemolysis on blood agar containing rabbit blood compared to sheep blood could be observed for A. haemolyticum, less pronounced for A. hippocoleae and A. pluranimalium. A synergistic hemolytic reaction with staphylococcal beta-hemolysin appeared to be constantly visible for A. hippocoleae, A. pluranimalium and A. pyogenes, with Streptococcus agalactiae for A. phocae and A. haemolyticum, with Rhodococcus equi for A. phocae, A. haemolyticum, A. pluranimalium and A. pyogenes and with A. haemolyticum for A. hippocoleae, A. pluranimalium and A. pyogenes, respectively. A reverse CAMP-reaction in the zone of staphylococcal beta-hemolysin could be observed for A.phocae and A.haemolyticum. In addition, a novel CAMP-like reaction could be noted between Psychrobacter phenylpyruvicus, identified by 16S rDNA sequencing, and A. phocae and A. haemolyticum. These synergistic or antagonistic hemolytic properties could possibly be used as additional criteria for identification of bacteria of genus Arcanobacterium.

Hyaluronic acid is thought to be one of the critical virulence factors of Streptococcus equi subsp. zooepidemicus. The present study was designed to study the role of hyaluronic acid capsular material in mediating adherence and to resist the phagocytosis of the host's immune defence. The studies were performed with two encapsulated S. equi subsp. zooepidemicus and two unencapsulated phase variants. The bacteria had been previously isolated from diseased pigs and monkeys in Indonesia. The presence of capsular material was determined using the hyaluronic acid decapsulation test and by electron microscopic studies. Both encapsulated bacteria showed mucoid colonies after cultivation on blood agar, grew with diffuse colonies in soft agar media and reacted negatively in the salt aggregation test. The unencapsulated bacteria grew with small colonies on blood agar, formed compact colonies in soft agar media and reacted positively in the salt aggregation test. Adherence and phagocytosis studies revealed that the encapsulated bacteria adhered significantly more to HeLa cells and were less phagocytosed by murine macrophages compared to unencapsulated bacteria. Pretreatment of the HeLa cells using hyaluronic acid or pretreatment of the bacteria by hyaluronidase decreased the adherence value of encapsulated bacteria. Pretreatment of bacteria with pronase had no effect. The presented results strongly indicate that the hyaluronic acid capsular material contributes to adherence properties of S. equi subsp. zooepidemicus and might help the bacteria to resist phagocytosis by macrophages.

The present study was designed to identify and comparatively investigate 35 beta-hemolytic streptococci isolated from stranded harbor porpoises or from animals caught in fishing nets of the North and Baltic seas. According to biochemical and serological data and to lectin agglutination tests with the lectin of Arachis hypogaea , all 35 isolates could be classified in Lancefield’s serological group L and could be identified as Streptococcus dysgalactiae subsp. dysgalactiae . All 35 group L streptococci were uniformly sensitive to most of the antibiotics tested. To further analyze the epidemiological relationship, the isolates were subjected to macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis. Digestion of the chromosomal DNA with the restriction enzymes Sma I and Apa I revealed that most of the group L streptococci seemed to be apparently identical or related. These results indicate that one clone or at least related group L streptococcal clones play an important role for infections of harbor porpoises of the North and Baltic seas. This might possibly be caused by a direct transfer of the bacteria from animal to animal.

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-finger-printing. 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.

Summary In the present study 70 Streptococcus porcinus isolates could be identified and further characterized by cultural and biochemical properties, by determination of their antibiotic susceptibility and by serological classifications. The S. porcinus included serogroup- and serotype-reference strains, presumptive group candidates and isolates obtained from routine diagnostics. All cultures investigated appeared with a broad zone of β-haemolysis on sheep blood agar showed a CAMP-like reactivity in the zone of staphylococcal β-lysin and had the typical biochemical properties of this species. Determination of antibiotic susceptibility revealed a high number of cultures to be susceptible to ampicillin, bacitracin, chloramphenicol, penicillin and vancomycin. Resistances could be observed for erythromycin, minocycline, sulfamethoxazol-trimethoprim, streptomycin and tetracycline. Serogrouping and serotyping could be performed with autoclaved extracts of the bacteria and group- and type-specific antisera prepared against reference strains and group candidates by immunodiffusion reactions. By serogrouping almost all cultures could be classified into serogroup E, U, V or P. Some group E streptococci could additionally be serotyped with type II, VI, VII and group X specific antiserum indicating that group X represents an additional type antigen of serogroup E. None of the antigen preparations reacted with serotype IV, V or group candidate NG1 specific antiserum. The described properties might help to identify and further characterize isolates of the species. S. porcinus, possibly useful in epidemiological aspects.

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.

Comparable to previously conducted phenotypical and genotypical investigations characterizing Arcanobacterium canis, a newly described species with the type strain A. canis DSM 25104 isolated from an otitis externa of a dog, four additional A. canis strains isolated from infections of three dogs and one cat could reliably be identified by phenotypic properties, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and by sequencing the genomic targets 16S rDNA, 16S-23S rDNA intergenic spacer region, 23S rDNA, and the genes rpoB and gap. All four A. canis investigated in the present study were isolated from the infected animals together with several other bacterial species indicating that the pathogenic importance of A. canis remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approaches might help to identify A. canis in future and might elucidate the role this species plays in infections of dogs and cats.

The present study was designed to comparatively investigate 34 beta-hemolytic streptococci isolated from infected pigs and monkeys from various islands in Indonesia. According to the serological and biochemical data, all 34 isolates were Lancefield's serological group C streptococci and could be identified as Streptococcus equi subsp. zooepidemicus. Of the 34 group C streptococci investigated, 28 grew on solid media in large, mucoid colonies, in fluid media at a uniform turbidity, and in soft agar in diffuse colonies. A decapsulation test with a hyaluronidase-producing Staphylococcus aureus strain revealed the hyaluronic acid nature of the capsular material. The remaining six streptococci grew on solid media in small, nonmucoid colonies, in fluid media as sediment with clear supernatant, and in soft agar in compact colonies. Determination of surface hydrophobicity by salt aggregation revealed a hydrophilic surface for the encapsulated bacteria and a hydrophobic surface for the unencapsulated group C streptococci. To further analyze the epidemiological relationships, all 34 mucoid and nonmucoid isolates from pigs and monkeys were subjected to protein and DNA fingerprinting. The latter was performed by pulsed-field gel electrophoresis. The protein profiles of all 34 isolates and the DNA profiles of 32 isolates appeared to be identical, with the DNA profiles of 2 isolates being closely related, indicating that a single virulent clone is responsible for this disease outbreak in Indonesia.

Summary Immunoglobulin G (IgG) binding proteins on the surface of Streptococcus suis could be readily detected by direct cultivation of the bacteria on nitrocellulose membranes and subsequent treatment of the membranes with human IgG. Among the 75 S. suis isolates tested two cultures (S. suis P43, S. suis P143) caused a blue colouration of the membranes indicating IgG binding activities. The IgG binding proteins could be solubilized by heat treatment of the bacteria at an acid pH and also by mutanolysin treatment. Western blot analysis revealed numerous protein bands with IgG binding activities. The IgG binding proteins were also released into the culture supernatant of the bacteria. This could be detected for 51 of the 75 S. suis using a microfiltration assay. In binding studies with 125I-IgG S. suis P43 and S. suis P143 but none of the other S. suis isolates showed a significant binding of the protein. These two cultures additionally bound 125I-albumin, 125I-α2-macroglobulin and 125I-fibrinogen all from humans but not 125I-chicken IgG or 125I-human haptoglobin 2–1. The binding profiles of the two S. suis cultures tested indicate a close relation of these binding proteins with streptococcal protein G.

A haemolysin produced by Actinomyces pyogenes ATCC 8164 was purified from culture supernatant by ammonium sulphate and polyethylene glycol precipitation, ion-exchange chromatography on DEAE-Sephacel, and fast-protein-liquid-chromatography on Superose 12 prep grade. The purified haemolysin, designated as pyolysin, displayed a single band on poly-acrylamide gel electrophoresis, indicating a molecular weight of 55000. Additionally, using gel filtration, the same molecular weight was estimated. Further studies of the eluate of ion-exchange chromatography using isoelectric focusing also revealed a single protein band at pH 9.38 with haemolytic activity. A specific antiserum produced against pyolysin inhibited the haemolytic activity. The purity of the isolated protein was also determined by Western Blot analysis with antiserum obtained from a cow inoculated with culture supernatant from A. pyogenes and Peptococcus indolicus. The isolated pyolysin appeared to be heat-labile and displayed cytotoxic effects on poly-morphonuclear leucocytes and on pTK2 kidney cells.

Summary In this study, selected haemagglutination-positive and haemagglutination-negative streptococci of serological group B were investigated in HeLa-cell- and alveolar-macrophage adherence tests. The cultures, isolated from bovine milk samples, had been previously serotyped and further characterized. All haemagglutination-positive group B streptococci adhered in high numbers to HeLa cells and alveolar-macrophages of rabbits. Haemagglutination-negative group B streptococci showed no comparable adherence. The adherence to alveolar-macrophages of rabbits was further characterized. This adherence appeared to be time- and temperature-dependent and could be inhibited by heat or proteolytic treatment of the bacteria. After opsonization of the bacteria, an adherence to alveolar-macrophages could also be observed for haemagglutination-negative group B streptococci, indicating a separate adherence mechanism. The group B streptococcal adherence might represent an important prerequisite for invasion and possibly intracellular survival of this bacterial species.

In this study, 133 streptococci of serological group B, which were isolated from bovine milk samples in Thuringia and Hesse, were serotyped and further characterized. Serotyping of 99 group B streptococci from Thuringia revealed mainly the antigen patterns III, IV, IV/X and NT/X, and serotyping of the cultures from Hesse revealed mainly the antigen patterns IV, IV/X and NT/c alpha. The occurrence of polysaccharide- and protein-type antigens appeared to be related to various other characteristics of the bacteria. A significant relationship was observed among serotype, growth properties of the bacteria in fluid media and soft agar, and the occurrence of pigmentation. In addition, fibrinogen-binding properties of the bacteria and the ability to agglutinate rabbit erythrocytes seemed to be related to specific serotypes of the group-B streptococci. All these data were used to characterize group-B streptococcal isolates individually. This could be of importance in epidemiological studies and might help to explain the process of infection with this micro-organism.

In this study, all 88 streptococci of serological group L isolated from cows, pigs, poultry and humans bound 125I-immunoglobulin G, and, in addition, 22 cultures interacted with 125I-albumin. IgG- and albumin-binding sites were solubilized from the streptococcal surface by heat extraction at an acid pH and also by mutanolysin treatment of the bacteria. Western blot analysis of these binding proteins revealed that almost identical protein bands were responsible for 125I-IgG and -albumin binding. Certain protein fractions of the cultures interacted exclusively with 125I-IgG, indicating that there are two groups of IgG receptors among streptococci of this serogroup.

Different pathogens cause both clinical and subclinical mastitis and Staphylococcus aureus is the most common. One hundred and thirty six apparently healthy cows from six Local Government Areas of Plateau state, Nigeria were sampled for the isolation of this pathogen. Three hundred and thirty nine quarter milk samples from the cows were collected, out of which 102 S. aureus were isolated. Twenty isolates were further analysed at molecular level. The species of the 20 strains were confirmed by PCR amplification using S. aureus specie-specific primers derived from the 23S rDNA and by amplification of nuc, coa and spa genes. Epidemiological relationships of the strains were studied by macro-restriction analysis of their chromosomal DNA using pulse field gel electrophoresis (PFGE). Among the 20 S. aureus strains identified, PFGE revealed an identical DNA pattern for 18 strains while two strains differed in two bands. These differences were revealed by the amplification of the spa gene. The relationship between the S. aureusisolated from nomadic raised cows discovered in the study areas remains unclear as only two pulse types were observed. Key words: Staphylococcus aureus, subclinical mastitis, pulsed-field gel electrophoresis, macro-restriction.

Summary In the present study, 17 Rhodococcus equi isolates from animals and 13 R. equi isolates from humans were correctly identified by the use of the API Coryne-test system. The biochemical characteristics revealed no significant differences between isolates from animals and humans. Heat extractions at acid pH or autoclave extractions of the bacteria and the use of monospecific antisera against type antigens 1–7 allowed the serotyping of the bacteria. The majority of the R. equi isolates reacted with serotype 1 and serotype 2-specific antisera, extracts of two human isolates reacted with serotype 5 and serotype 6-specific antisera, respectively. The determination of biochemical and serological properties of R. equi might help to characterize individual cultures of this species. This might be useful in epidemiological studies.

All 83 bacterial strains isolated from seven farms in three areas of the island of Java in Indonesia investigated in the present study could be identified as Streptococcus agalactiae. Identification was performed by cultural, biochemical and serological properties and by polymerase chain reaction amplification of species-specific parts of the gene encoding the 16S rRNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor (cfb) gene. All isolates were unpigmented. almost all of the isolates had the serotype pattern II/X. Despite these similarities a macrorestriction analysis of the chromosomal DNA of the bacteria revealed no significant homologies of the DNA-fingerprints of the S. agalactiae from the various areas. This last finding might possibly indicate that a single ancestral unpigmented serotype II/X S. agalactiae clone was responsible for the mastitis situation on Java and had evolved separately in the various farms and regions.

In the present study matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for species identification of 98 bacteria previously classified phenotypically and genotypically to genera Arcanobacterium and Trueperella . Species identification was carried out by comparing the main spectra of each strain with the main spectra of reference strains of both genera and 3740 database entries included in the MALDI Biotyper 2.0 software package (Bruker Daltonik GmbH, Bremen, Germany). MALDI-TOF MS correctly identified (log (score) values ≥ 2.0) all investigated strains of the species A. ( T. ) bialowiezense ( n = 3), A. ( T. ) bonasi ( n = 7), A. haemolyticum ( n = 10), A. pluranimalium ( n = 1) and A. ( T. ) pyogenes ( n = 77). According to the present results MALDI-TOF MS had a comparable discriminating power than previously conducted tests on DNA level. Further studies with strains isolated from human infections would show the robustness of MALDI-TOF MS for identification of bacteria of these genera.

Summary Most of the 150 Streptococcus suis isolates from pigs and ruminants used in this study grew under aerobic conditions and were α-hemolytic on sheep blood agar. Part of the cultures required an increased CO 2 concentration. These cultures, representing the CO 2 -dependent ecovar of S. suis , were mainly β-hemolytic on sheep and horse blood agar and gave a synergistic hemolytic reaction with staphylococcal β-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 mannitolpositive 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.

Summary It has been 16 years since the identification of S. intermedius as a new species. Numerous investigations using cell wall and DNA analytic methods have now clearly demonstrated significant differences to warrant the creation of this new species. However, studies investigating virulence factors associated with S. intermedius have not uncovered evidence that differentiates virulent from non-virulent isolates. Therefore, at the present time, it is difficult for veterinary clinicians and microbiologists to determine the clinical significance of many S. intermedius isolates. Host-bacterial interactions and the hosts' immune status appear to be the factors most crucial in determining the outcome of infections, not the virulence of the organism. Continued research in the virulence factor field will hopefully lead to a better understanding on how we can differentiate virulent from non-virulent isolates of S. intermedius.

The present study was designed to characterize phenotypically and genotypically nine Arcanobacterium abortisuis strains collected from specimen of pigs in a period of nine years. All nine A. abortisuis strains and A. abortisuis reference strain DSM 19515 displayed a synergistic hemolytic reaction with Staphylococcus aureus β-hemolysin, Rhodococcus equi, and Arcanobacterium haemolyticum indicator strains and showed the typical biochemical properties of this species. The species identity could be confirmed by identification and sequencing of the 16S-23S rDNA intergenic spacer region (ISR), which appeared to be a useful target for genotypic characterization of this bacterial species. The A. abortisuis strains of the present study were isolated from specimen of pigs together with various other bacterial species indicating that the pathogenic importance of this newly described species remains to be elucidated.

Serotyping of 103 streptococci of serological group B isolated from human clinical specimens in Indonesia revealed mainly type pattern Ia/c and type patterns III and V, either alone or in combination with protein antigens c or R. Few cultures had serotype II and IV, none of the cultures had type antigen Ib or X. Extracts of one group B streptococcal culture reacted with antibodies specific to type antigens II, IV and R and one culture with antibodies specific to candidate type 7271. Protein antigen c was present as c alpha, c alpha, beta but not as c beta component. Group B streptococci with type antigen R grew generally as a granular sediment with clear supernatant, formed compact colonies in soft agar and reacted positively in the salt aggregation test. Group B streptococci without surface antigen R grew mainly with a uniform turbidity of the growth media, formed diffuse colonies in soft agar and were negative in the salt aggregation test.

Summary 125-I-IgG binding activities were observed with 15 (17 %) of 90 S. intermedius isolates from dogs and 39 (95%) of 41 S. hyicus isolates from pigs. Binding activities were not detected with S. hyicus isolates from cows. The IgG binding proteins of 2 S. intermedius, 2 S. hyicus, and protein A from S. aureus Cowan I were isolated from their cell surfaces. The proteins precipitated with IgG preparations from human, rabbit, pig, dog and horse, but not with IgG from cow, mouse and chicken. This indicated that these IgG binding proteins could be classified as type I receptors. In addition, the isolated proteins from all 3 staphylococcal species precipitated with polyclonal chicken anti-protein A antiserum. SDS-PAGE, Western blotting and gel isoelectric focussing of the proteins revealed numerous bands in the 42,000 D range and acid isoelectric points. The isoelectric point of the isolated proteins from both S. intermedius cultures was slightly more acidic than those from S. hyicus and S. aureus. The present results indicate a close functional and antigenic similarity, if not identity, between IgG binding proteins of S. intermedius and S. hyicus, and protein A of S. aureus.

Cell surface hydrophobicities of Actinomyces pyogenes were determined by measuring the adherence of the bacteria to hexadecane droplets and by salt aggregation tests. Among 42 A. pyogenes cultures tested 25 (60%) adhered strongly (adherence greater than or equal to 75%) and 17 (40%) less pronounced (adherence between 25-75%) to the hexadecane droplets. Pre-treatment of the bacteria with proteolytic enzymes completely eliminated the adherence properties whereas heat treatment had no effect. The salt aggregation studies revealed that 4 (10%) cultures aggregated in ammonium sulfate solutions of a molarity of 0.05 mol/l, 5 (12%), 14 (33%) and 3 (7%) cultures in ammonium sulfate solutions with molarities of greater than or equal to 1.5 mol/l, greater than or equal to 3 mol/l and greater than or equal to 4.5 mol/l, respectively. No aggregation at all could be observed with 16 (38%) of the cultures. Pronase treatment completely eliminated the salt aggregation reactions, trypsin- and heat treatment had no effect. The results from hexadecane adherence and salt aggregation did not correspond. The differences in surface hydrophobicities, possibly related to adherence properties of A. pyogenes, could be used for epidemiological typing of individual cultures of this bacterial species.

Summary The group B streptococcal surface antigens X and R were isolated from culture supernatant by ammonium sulphate precipitation and subsequent high performance gel filtration (HPLC). The type antigens appeared in numerous protein bands. Type antigen X was in the range of 180 kD, type antigen R, in the range of 116 kD. The protein antigens reacted specifically with their homologous antisera in Western blot analysis and in immunoelectrophoresis, indicating that there was no immunological relationship between both proteins. Preparative isoelectric focusing of the X and R antigens revealed for both proteins isoelectric points at acid pH. Specific antibodies produced against the purified proteins could be used for routine serotyping without further absorption.

The ATB 32 Staph system correctly identified 45 of 54 Staphylococcus hyicus cultures isolated from pigs and cattle. The biochemical profiles of the remaining nine cultures were not listed in the product data base. The 40 porcine and 14 bovine cultures resulted in three and seven different biochemical profiles, respectively. In parallel experiments, almost all S. hyicus cultures showed hemolytic reactions on chocolate agar, had CAMP-like reactivities in the zone of lysis of the staphylococcal beta-lysin, and had bacteriolytic properties on Micrococcus luteus cells. In addition, the S. hyicus cultures were unpigmented, were DNase positive, expressed an S. hyicus-specific teichoic acid, and were usually coagulase positive in porcine plasma. Most of the porcine strains were protein A positive, and two porcine cultures were clumping factor positive.

Summary Staphylococcus chromogenes and Staphylococcus hyicus showed bacteriolytic activities towards a Micrococcus luteus reference strain. This was demonstrated on tryptone soya agar containing M. luteus cells. Both bacteriolytic enzymes could be isolated by ionic exchange chromatography and subsequent gel filtration. The isolated bacteriolysin of S. chromogenes lysed the M. luteus reference culture, was heat inactivated by 95°C and precipitated specifically with antiserum produced against the bacteriolysin of S. hyicus.

The Strep-Zym identification system, a combination of 23 enzymatic tests, allowed a rapid biochemical characterization of Streptococcus uberis. The biochemical profiles of the S. uberis cultures clearly differed from those of S. agalactiae and S. dysgalactiae. Serological grouping of S. uberis revealed polysaccharide antigens of groups E, G, P and U. Some cultures of S. uberis demonstrated CAMP-like synergistic hemolytic activities on sheep blood agar and reacted specifically with the lectins of Helix pomatia and Dolichos biflorus. The occurrence of group polysaccharides, CAMP-like reactivities, and the lectin agglutination reactions were obviously not related to each other or to any of the biochemical properties. These reactions, possibly of importance as virulence factors, might serve as epidemiological markers.

Summary Purification of a murine IgG3 monoclonal antibody (Mab 22) directed against an epitope of Chlamydia-specific lipopolysaccharide by affinity chromatography on recently described solid phase IgG Fc-receptors from Streptococcus dysgalactiae is reported. SDS-PAGE studies revealed the purity of the eluted antibody. The purified Mab 22 was characterized by determination of class, subclass and light chain-type, and by dot tests and immunoblot analysis.

Summary Streptococcal group-specific antigens and B-streptococcal type antigens were solubilized with a murolytic enzyme from Staphylococcus hyicus. This facilitated the effective serogrouping and serotyping of streptococci isolated from humans and various animals. Zusammenfassung Ablosung von gruppen- und typenspezifischen Streptokokkenantigenen mit einem murolytischen Enzym von Staphylococcus hyicus Das murolytische Enzym vom Staphylococcus hyicus fuhrte zu einer Freisetzung von gruppen-spezifischen Streptokokkenantigenen und von B-Streptokokken-Typenantigen. Dies ermoglichte eine erfolgreiche Gruppenbestimmung und Serotypisierung von Streptokokken isoliert von Mensch und verschiedenen Tierarten.