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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.

An increase in prevalence of multidrug-resistant Acinetobacter spp. in hospitalized animals was observed at the Justus-Liebig-University (Germany). Genotypic analysis of 56 isolates during 2000–2008 showed 3 clusters that corresponded to European clones I–III. Results indicate spread of genotypically related strains within and among veterinary clinics in Germany.

Zusammenfassung In den vorliegenden Untersuchungen wurde gepruft, ob und inwieweit die Firefly-Biolumineszenz uber die Bestimmung von bakteriellem ATP zur Ermittlung der Sensibilitat bzw. Resistenz von Bakterienstammen gegenuber verschiedenen Antibiotika herangezogen werden kann. Die Versuchsansatze erfolgten in flussigen Medien, die mit den jeweiligen Testbakterienstammen beimpft wurden und Gentamycin, Polymyxin B, Tetrazyklin oder Chloramphenicol als Antibiotikum enthielten. Nach einer Inkubation von drei Stunden bei 37°C wurde das bakterielle ATP gemessen und mit den an einer Antibiotika-freien Bouillon ermittelten Werten sowie mit den entsprechenden Hemmhof-Durchmessern in der herkommlichen Sensitivitatstestung (Agardiffusionstest) verglichen. Die bisherigen, an Escherichia coli- und Staphylococcus aureus-Stammen sowie an s-hamolysierenden Streptokokken, Pseudomonas aeruginosa und Proteus sp. erhobenen Befunde haben gezeigt, das mit dem Biolumineszenz-Verfahren schon nach einer dreistundigen Inkubation signifikante Unterschiede im ATP-Gehalt empfindlicher und resistenter Bakterienstamme festzustellen sind, welche als Ausdruck einer gehemmten bzw. mehr oder weniger ungehemmten Vermehrung der Bakterien im Versuchsansatz wahrend des Inkubationszeitraumes angesehen werden mussen. Die Resultate korrelierten gut mit jenen des Agardiffusionstestes. Die Methode ermoglicht nach den bisherigen Erfahrungen eine schnelle Bestimmung des Resistenzverhaltens von Infektionserregern aus klinischem Material und damit die unverzugliche Einleitung einer gezielten antibakteriellen Behandlung besonders bei Risikopatienten. Daruber hinaus ergaben sich bei manchen Bakterienstammen, die im Agardiffusionstest lediglich als resistent erkannt wurden, aufgrund des weiten Mesbereichs des Photomultipliers Hinweise auf eine partielle Wachstumhemmung der Bakterien durch das verwendete Antibiotikum. Summary Resistance testing of bacteria by firefly bioluminescence A rapid test Studies were made to determine whether and to what extent firefly bioluminescence can be used to determine bacterial ATP for measurement of sensitivity or resistance of bacterial strains to different antibiotics. The experiments were carried out in fluid media inoculated with the test bacterial strain and containing gentamycin, polymyxin B, tetracycline or chloramphenicol as antibiotics. After incubation for 3 hours at 37°C the bacterial ATP was measured and compared with the amount in an antibiotic-free broth; at the same time the inhibition zone diameters in the sensitivity test (agar diffusion test) were compared. Tests on E. coli and Staph. aureus strains as well as s-haemolytic streptococci, Pseudomonas aeruginosa and Proteus sp. showed that already after 3 hours incubation the bioluminescence test revealed significant differences in ATP content between sensitive and resistant strains; this represented an inhibited or a more or less uninhibited multiplication of the bacteria in the material during the incubation period. The results correlated well with those with the agar diffusion test. The method allows rapid determination of resistance status of infective agents isolated from clinical material and thus supplies an opportunity for prompt antibacterial therapy of patients at risk. In addition, it was found that many bacterial strains which the agar diffusion test recognized as resistant revealed by measurements of the photomultiplier evidence of partial growth inhibition of the organism by the antibiotic in use. Resume Epreuve de resistance des bacteries au moyen de la “Firefly”-bioluminescence Un test rapide On a examine dans cette recherche si et dans quelle mesure la “Firefly”-bioluminescence pouvait etre utilisee pour obtenir la sensibilite et respectivement la resistance de souches bacteriennes vis-a-vis de differents antibiotiques par la determination de l'ATP bacterienne. Les recherches ont ete effectuees dans de milieux liquides inocules avec les differents souches bacteriennes test et contenant de la gentamycine, polymyxine B, tetracycline et du chloramphenicol. Apres une incubation de 3 heures a 37°C, l'ATP bacterienne a etemesuree et comparee avec des valeurs d'un bouillon sans antibiotique et avec la mesure de la zone d'inhibition dans le test courant de diffusion en gelose (test de diffusion sur agar). Les resultats trouves jusqu'a maintenant sur des souches de E. coli, Staph. aureus, Sc. beta-hemolytique, Pseudomonas aeruginosa et Proteus sp. ont montre que l'on pouvait etablir en bioluminescence apres trois heures d'incubation deja des differences significatives dans le taux d'ATP des souches bacteriennes sensibles et resistantes qui pouvaient etre vues comme l'expression d'une multiplication inhibee ou plus ou moins inhibee des bacteries dans le milieu d'essai durant le temps d'incubation. Les resultats ont eu une bonne correlation avec ceux du test de diffusion sur agar. La methode rend possible selon les resultats trouves une determination rapide du comportement de resistance des germes infectieux a partir d'un materiel clinique et la conduite immediate d'un traitement antibacterien approprie en particulier chez des patients a haut risque. Des mises en evidence d'une inhibition partielle de croissance des bacteries avec l'antibiotique utilise ont ete revelees au moyen du photomultiplicateur chez plusieurs souches de bacteries qui avaient ete reconnues uniquement resistantes au test de diffusion sur agar. Resumen Prueba de la resistencia de las bacterias mediante la bioluminiscencia FIREFLY Una prueba rapida En los estudios presentes se examino si y en que grado se puede recurrir a la bioluminiscencia Firefly encaminada a determinar la ATF bacteriana para perquirir la sensibilidad resp. la resistencia de las estirpes bacterianas frente a los distintos antibioticos. Las siembras se efectuaban en medios liquidos, los cuales habian sido inoculados con las estirpes bacterianas de prueba junto con gentamicina, polimixina B, tetraciclina o cloramfenicol como antibiotico. Tras la incubacion a 37°C durante tres horas, se midio la ATF bacteriana, comparandose con los valores obtenidos en un caldo sin antibioticos y con los diametros de las zonas de inhibicion correspondientes en la prueba de sensitividad convencional (prueba de difusion sobre gelosa). Los hallazgos evidenciados hasta ahora en cepas Escherichia coli, Staphylococcus aureus, estreptococos hemolizantes s, Pseudomonas aeruginosa y Proteus sp. senalaron que con el procedimiento de la bioluminiscencia ya se pueden poner en evidencia, tras una incubacion de tres horas, diferencias significantes en el contenido de ATF de las estirpes bacterianas sensibles y resistentes, las cuales se tienen que considerar como expresion de una multiplicacion inhibida resp. mas o menos libre de bacterias en el enunciado del experimento durante el espacio de tiempo que duro la incubacion. Los resultados correlacionaban bien con los de la prueba de difusion en agar. La tecnica permite, segun los conocimientos actuales, establecer una determinacion rapida del porte de la resistencia de los agentes etiologicos del material clinico y, con ello, la induccion inmediata de un tratamiento antibacteriano dirigido sobre todo en los pacientes en peligro. Mas alla de esto, se infirieron en algunas estirpes bacterianas, las cuales se reconocieron solo en la prueba de difusion en gelosa como resistentes, a la vista del ambito amplio de medida del fotomultiplicador, indicios en cuanto a una inhibicion parcial del crecimiento de las bacterias por el antibiotico utilizando.

Zusammenfassung 114 Staphylokokken-Kulturen, nach biochemischen Gesichtspunkten in 16 Staphylococcus (S.) aureus-, 87 S. intermedius- und 11 Koagulase-negative Staphylokokken-Stamme differenziert, wurden mittels eines Kaninchen-IgG-Enzym-Konjugates auf ihr Protein-A-Bildungsvermogen untersucht. Dabei erwiesen sich 29 Stamme (14 × S. aureus, 15 × S. intermedius) als Bildner von zellgebundenem Protein A. Demgegenuber lies sich bei 88 Stammen (15 × S. aureus, 72 × S. intermedius, 1 × Koagulase-negative Staphylokokken) Protein A im Kulturfiltrat nachweisen. Die hochsten Extinktionen wurden in diesem Enzym-“Immun”-Test mit S. aureus-Kulturen erreicht, wahrend die S. intermedius-Stamme uberwiegend niedrigere Enzymreaktionen auslosten. Auf die Bedeutung von Protein-A-bildenden Staphylokokken als Ursache von Fehlbeurteilungen in immunologischen Testsystemen (z. B. Immunfluoreszenz) wird hingewiesen. Summary A simple ELISA-technique for detection of protein A-producing staphylococci The protein A-forming capacity of 114 Staphylococcus (S.) aureus-cultures (16 S. aureus, 87 S. intermedius and 11 coagulase-negative S.-strains) was determined by an enzyme-“immuno”-assay using a rabbit-IgG-peroxidase-conjugate. Cell-bound protein A could be demonstrated in 29 strains (14 × S. aureus, 15 × S. intermedius), whereas 88 strains (15 × S. aureus, 72 × S. intermedius, 1 × coagulase-negative S.-strain) showed production of extracellular protein A. S. aureus-strains yielded the highest extinctions in this assay, contrary to the S. intermedius-cultures, which predominantly produced low enzyme-reactions. The authors discuss the significance of protein A-producing staphylococci causing false-positive results in immunological assays (e. g. immunofluorescence). Resume Mise en evidence de la formation de la proteine A des staphylocoques avec la technique ELISA On a recherche au moyen d'un conjugue enzymatique IgG-lapin les possibilites de formation de la proteine A dans 114 cultures de staphylocoques, differencies biochimiquement en 16 Staphylococcus aureus, 87 S. intermedius et 11 souches de staphylocoques coagulase-negative. 29 souches (14 × S. aureus et 15 × S. intermedius) produisaient une proteine A liee a la cellule. Une proteine A a ete mise en evidence dans un filtrat de culture chez 88 souches (15 × S. aureus, 72 × S. intermedius et 1 × staphylocoques coagulase-negative). Les plus fortes extinctions ont ete obtenues dans ce test enzymatique “immun” avec des cultures S. aureus, alors que les souches S. intermedius declenchaient des reactions enzymatiques plus faibles. La signification des staphylocoques producteurs de proteine A comme cause d'erreurs d'interpretation dans des systemes de test immunologiques (p. ex. immunofluorescence) est evoquee. Resumen La puesta en evidencia de la formacion de proteina A por los estafilococos mediante la tecnica del ELISA 114 cultivos de estafilococos, diferenciados con arreglo a puntos de vista bioquimicos en 16 estirpes S. aureus, 87 S. intermedius y 11 coagulasa-negativas, se examinaron mediante un conjugado de IgG de conejo-enzima en cuanto a su potencia de formacion de proteina A. Aqui se manifestaron 29 estirpes (14 × S. aureus, 15 × S. intermedius) como formadoras de proteina A ligada a la celula. Frente a esto, se pudo poner en evidencia en 88 estirpes (15 × S. aureus, 72 × S. intermedius, 1 × estafilococos coagulasa-negativos) proteina A en el filtrado del cultivo. Las extinciones maximas fueron conseguidas en esta prueba de enzima «inmunitaria» con S. aureus, mientras que las estirpes S. intermedius desencadenaban reacciones enzimaticas mas bajas. Llaman los autores la atencion sobre la significancia de los estafilococos productores de proteina A como causa de resultados positivos falsos en sistemas de pruebas inmunologicas (por ej. inmunofluorescencia).

Zusammenfassung Insgesamt 179 Kot- und Darminhaltsproben von Hunden wurden vergleichend bakteriologisch und virologisch untersucht. Aus 108 Proben von Hunden (Einzeltiere) mit schweren Enteritiden gelang 22mal die Isolierung von C. f. subsp. jejuni und 46mal der Parvovirusnachweis. In 13 Fallen lagen beide Erreger gemeinsam vor. Bei 54 Kontrollproben gesunder Hunde konnte C. f. subsp. jejuni in 1 Fall und Parvovirus in 4 Fallen isoliert werden. 15 von 17 Hunden einer Versuchstierzucht, in der akute Gastroenteritiden aufgetreten waren, erwiesen sich ebenfalls als Trager von Campylobacter-Keimen. Hier gelang der Parvovirusnachweis in 4 Fallen, davon 3mal gemeinsam mit C. f. subsp. jejuni. Die Ergebnisse lassen den Schlus zu, das sowohl C. f. subsp. jejuni als auch Parvovirus eine eigenstandige atiologische Rolle bei akuten Gastroenteritiden des Hundes spielen. Der auffallend gehaufte gleichzeitige Nachweis beider Erreger bei schwer erkrankten Tieren spricht daruber hinaus fur ein synergistisches Zusammenwirken. Summary Aetiological significance of Campylobacter fetus subsp. jejuni and Parvovirus for acute enteritis in dogs A total of 179 faecal and gut contents samples from dogs (individual animals) were compared by bacteriological and virological examination. From 108 samples from dogs with severe enteritis C. f. subsp. jejuni was isolated 22 times and Parvovirus 46 times. In 13 samples both agents were found. With 54 control samples from healthy dogs C. f. subsp. jejuni was isolated in one case and Parvovirus in four. 15 of 17 dogs in a breeding kennels where acute gastroenteritis had occurred were found to be carriers of Campylobacter. Here the isolation of parvovirus succeeded in 4 cases, in three of which C. f. subsp. jejuni was also isolated. The results lead to the conclusion that both C. f. subsp. jejuni and Parvovirus play an aetiological role in acute canine gastroenteritis. The frequent simultaneous isolation of both agents in severely sick animals also suggests a synergistic effect. Resume A propos de la signification etiologique de Campylobacter fetus subsp. jejuni et Parvovirus dans des enterites aigues du chien 179 echantillons de matieres fecales et de contenu intestinal ont ete examines en comparaison bacteriologiquement et virologiquement. Sur 108 echantillons provenant de chiens atteints d'une grave enterite, il a ete possible d'isoler C. F. ssp. jejuni 22 fois et de mettre en evidence un Parvovirus 46 fois. On a rencontre les deux agents dans 13 cas. C. f. ssp. jejuni a ete isole dans 1 cas et Parvovirus dans 4 cas avec 54 echantillons de controle de chiens en bonne sante. 15 chiens sur 17 d'un elevage d'experience ayant presente une atteinte de gastroenterite aigue se sont reveles etre porteurs de Campylobacter. La mise en evidence dans ce cas de Parvovirus a ete possible dans 4 cas, dont 3 avec C. f. ssp. jejuni. Les resultats permettent de conclure qu'aussi bien D. v. ssp. jejuni que Parvovirus jouent un role etiologique independant lors de gastroenterites aigues du chien. La mise en evidence frequente et frappante des deux germes chez des animaux gravement malades parle pour une action commune synergique. Resumen Sobre la significacion etiologica de Campylobacter fetus subsp. jejuni y del Parvovirus para las enteritis agudas del perro Se examino de forma comparada bacteriologica y virologicamente un total de 179 muestras de estiercol y contenido intestinal de perros. De 108 muestras de perros (animales individuales) con enteritis graves se logro aislar 22 veces Campylobacter fetus subsp. jejuni y 46 veces la identificacion del Parvovirus. En 13 casos se hallaban juntos ambos agentes etiologicos. Entre 54 muestras de control de perros sanos pudo aislarse C. f. ssp. jejuni en 1 caso y Parvovirus en 4 ocasiones. 15 de 17 perros de una explotacion zootecnica experimental, en la cual habian aparecido gastroenteritis agudas, tambien resultaron ser portadoras de germenes Campylobacter. Aqui se logro identificar el Parvovirus en 4 casos, 3 de ellos junto con C. f. ssp. jejuni. Los resultados obtenidos admiten el que se saque en conclusion que tanto C. J. ssp. jejuni como Parvovirus juegan un papel etiologico independiente en las gastroenteritis agudas del perro. La puesta en evidencia simultanea, sorprendentemente tan acumulada, de ambos agentes etiologicos en los animales enfermos de gravedad aboga, ademas de esto, a favor de un concurso sinergistico.

In the present study 320 milk samples collected from 160 apparently healthy camels of three different locations in Sudan were investigated for the presence of Staphylococcus aureus resulting in the isolation of this bacterial pathogen from 28 milk samples from 24 camels. Twenty-five S. aureus were identified phenotypically and by PCR mediated amplification of species-specific genes or gene segments. Investigation of the S. aureus for toxinogenic potential revealed that three S. aureus strains were positive for the enterotoxin encoding gene sec and the genes seg, sei, sem, sen and seo, representing the egc gene cluster. In addition all 25 S. aureus were positive for the superantigen-like encoding gene ssl7 (set1). Partial sequencing of gene sec of the three S. aureus strains yielded an almost complete sequence identity to the sequence of the sec variant sec2. However, all three sec2 genes of the present study showed a deletion of one base causing a frame shift and a corresponding earlier stop codon. According to the present results, the raw camel milk collected from three locations in Sudan seems to be, at least at this stage, of minor importance as vector causing staphylococcal food poisoning.

The present study was designed to characterize phenotypically and genotypically seven Arcanobacterium haemolyticum strains obtained from infections of six horses. All seven strains showed the cultural and biochemical properties typical of A. haemolyticum and were susceptible to most of the antibiotics tested. The species identification could be confirmed by amplification and sequencing of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region and by PCR amplification of species-specific parts of the gene encoding phospholipase D in A. haemolyticum . Use of the latter could possibly improve future identification of this generally human pathogenic bacterial species which, according to the present results, seems to occur also in infections of horses.

Prevalence and incidence of foot rot disease in free-ranging and captive bovine wild ruminant populations are increasing worldwide. Even species in which the disease has not been described in the past are presently affected by the co-working pathogens Dichelobacter nodosus and Fusobacterium necrophorum. This paper discusses disease control measures and the expense for a successful treatment of affected populations of mouflon. The rationale and perspectives of treating foot rot disease in wild mouflons are discussed.

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.

Phenotypic properties and species-specific PCR tests based on the nuc gene of Staphylococcus intermedius and S. aureus, and a conserved region of 16S rDNA were used to identify 45 S. intermedius and four S. aureus isolated from samples of dogs during routine diagnostics. Four S. pseudintermedius strains used for control purposes reacted positively with the S. intermedius nuc PCR showing the close relationship between both species. Investigating the 45 S. intermedius and four S. pseudintermedius strains for the prevalence of the exfoliative toxin SIET encoding gene yielded the presence of the gene for 21 of the S. intermedius and two of the S. pseudintermedius strains. Partial sequencing of the toxin gene of a single S. intermedius strain and comparing this sequence with that obtained from GenBank revealed an almost complete identity. The presence of the exfoliative toxin gene could mainly be found among S. intermedius isolated from skin and wound infections and from otitis externa possibly indicating a role of this toxin for the clinical symptoms.

A species specific PCR test, based on manganese-dependent superoxide dismutase A encoding gene sodA, was developed for the identification of Staphylococcus hyicus, an important bacterial pathogen in pigs. The designed primers allowed a rapid and reliable identification of phenotypically characterized S. hyicus, isolated in Russia, Germany and Denmark. No cross reactivities could be observed investigating staphylococcal reference strains representing 18 different species and subspecies. The use of the described primers might improve a future diagnosis of this bacterial pathogen.

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.

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 .

Staphylococcus aureus septicaemia was diagnosed in a dead, stranded harbour porpoise from the German Baltic Sea and in a live harbour porpoise by-caught in inner Danish waters and taken into captivity. Lesions included pyogranulomatous myocarditis, necrotising suppurative bronchopneumonia, pyelonephritis, osteomyelitis and leptomeningitis, and abscesses in lymph nodes and skeletal muscles. The captive animal had fibrinous suppurative epicarditis and pyogranulomatous myocarditis with abscesses. In both animals the organism was suspected to have entered through skin lesions or via the respiratory tract.

In the present study four methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains isolated from a dog (n = 3) and the anterior nares of the dog owner (n = 1) were investigated by conventional and molecular methods. The species identity of the four S. pseudintermedius strains was confirmed by conventional methods, by PCR mediated amplification of S. intermedius/S. pseudintermedius specific segments of thermonuclease encoding gene nuc and by restriction fragment length polymorphism analysis of phosphoacetyltransferase encoding gene pta. Investigation of the four S. pseudintermedius for toxinogenic potential revealed that all four strains were positive for the exfoliative toxin encoding gene siet and the leukotoxin encoding genes lukS, lukF. The oxacillin and penicillin resistance of the four S. pseudintermedius strains could be determined by cultivation of the strains on oxacillin resistant screening agar base, ChromID MRSA Agar and Brilliance MRSA Agar and by multiplex PCR detecting the resistance genes mecA and blaZ. The genetic relatedness of the strains was studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). According to PFGE all four S. pseudintermedius strains represent an identical bacterial clone indicating a cross transmission between the dog and the dog owner.

The pathological, microbiological and serological findings in harbour porpoises hunted in Greenlandic waters were compared with the findings in animals accidentally caught in fishing gear in the German North Sea and Baltic Sea. The body condition of the Greenlandic animals was good, whereas nine of 23 German harbour porpoises were moderately to markedly emaciated. Both groups were infested with parasites. In the Greenlandic animals parasitism of the aural peribullar cavity with Stenurus minor, of the liver and pancreas with Orthosplanchnus mironovi, of the lungs with Halocercus species and of the subcutaneous and mammary tissue with Crassicauda species was generally associated with a mild inflammatory response. No diseases associated with bacteria were identified in any of the Greenlandic harbour porpoises. In the porpoises from the German North Sea and Baltic Sea, parasites were present in the aural peribullar cavity (S minor), liver (Campula oblonga), first and second gastric compartment (Anisakis simplex) and in the lungs (Pseudalius inflexus and Torynurus convolutus). Moderate to marked pulmonary parasitism and suppurative pneumonia, not observed in the Greenlandic porpoises, were present in 11 and 10, respectively, of the 23 German porpoises. The suppurative pneumonia was attributed to bacterial infection with beta-haemolytic streptococci and Escherichia coil var haemolytica. Four Greenlandic and 10 German porpoises had positive porpoise morbillivirus-specific antibody titres suggesting that the virus was circulating in both populations.

Between 1991 and 1996, necropsies were performed on 445 harbour porpoises (Phocoena phocoena), in various states of preservation, stranded on German coasts or accidentally caught by German fishermen. The animals originated from the North and Baltic Seas, and 133 were considered suitable for histopathological, immunohistochemical and microbiological examination. Most of the lesions in these 133 porpoises were caused by parasites, in particular in the respiratory tract, two-thirds of the animals exhibiting pneumonia associated with the parasites. Pneumonia was considered to be the cause of death in 46% of the stranded subadult and adult animals. The findings gave no evidence of any epidemic due to bacterial or viral infection. Bacteriological examination suggested that pneumonia was mainly caused by secondary bacterial infection and not by parasitic infestation alone. Beta-haemolytic streptococci were considered to be the main infectious agents. Morbillivirus antigen was not detected immunohistochemically.

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.

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.

A case of systemic mycosis due to a Rhizopus sp. infection is described in a dead-stranded, 10-yr-old, male harbor porpoise (Phocoena phocoena) found on the beach of Neustadt, Schleswig-Holstein on the Baltic Sea (Germany). At necropsy, granulomatous mycotic lesions in brain, lung, kidneys, testis, and draining lymph nodes were found. In addition, a focal ulcerative gastritis of the first stomach, due to a nematode infection, was present and is suspected to be the portal of entry for the fungus.

In the present study, Staphylococcus (S.) hyicus strains isolated in Russia (n = 23) and Germany (n = 17) were investigated for the prevalence of the previously described genes sheta and shetb. Sheta was detected in 16 S. hyicus strains. Sheta-positive strains were mainly found among strains isolated from exudative epidermitis, and frequently together with the exfoliative toxin-encoding genes exhD and exhC. Partial sequencing of sheta in a single S. hyicus strain revealed an almost complete match with the sheta sequence obtained from GenBank. None of the S. hyicus strains displayed a positive reaction with the shetb-specific oligonucleotide primer used in the present study. According to the present results, the exotoxin encoding gene sheta seems to be distributed among S. hyicus strains in Russia and Germany. The toxigenic potential of this exotoxin, which does not have the classical structure of a staphylococcal exfoliative toxin, remains to be elucidated.

Summary In order to establish defined immunological parameters for Q fever infection models, a microtitre enzyme-linked immunosorbent fluorescence assay (ELISA) was used for the first time to analyse the humoral immune response of Balb/cJ and C57BL/6J mice after experimental infection with Coxiella burnetii strain ‘Nine Mile’ in phase I. The experimental infection evoked a seroconversion in all mice within 10 days. Typically, the immune response measured against the whole-cell antigen showed an early increase of immunoglobulin (Ig) M followed by a later increase of the IgG subclasses. The IgA was low during the entire investigation period. Within the IgG subclasses only IgG2a and IgG2b gained higher values, whereby C57BL/6J mice produced high IgG2b titres and significantly lower IgG2a titres. In contrast, Balb/cJ mice developed IgG2a and IgG2b at equal levels. The use of partial antigens of C. burnetii demonstrated that the dominating IgG2b reaction of the C57BL/6J mice was directed against the lipopolysaccharide (LPS) of C. burnetii. This reaction was almost absent in Balb/cJ mice. In contrast, the SP27 protein antigen did not evoke different IgG2b reactions within the two breeds. No significant influence was observed within the two breeds in regard to sex or between hormone synchronized and non-hormone synchronized animals.

Mycobactin J-dependent mycobacterial isolates from sheep, goat, and cattle herds with Johne's disease in Morocco, South Africa, the United States, and Germany were tested for the repetitive insertion sequence IS900 of Mycobacterium paratuberculosis by PCR. The IS900 PCR target sequence was detected in 90 of 93 fecal culture isolates tested (96.8%). Restriction fragment length polymorphisms (RFLPs) and in vitro growth characteristics were studied in 46 of the IS900-positive isolates and in two bovine vaccine strains of M. paratuberculosis. Five different RFLP types were identified in PvuII digests of genomic DNA by Southern hybridization with a DNA probe specific for IS900. All isolates of M. paratuberculosis could be classified into two major clusters by their growth rates as well as the relatedness of their PvuII-RFLP hybridization patterns. All of the sheep isolates were classified into cluster I (extremely slow growth), while all cattle and goat isolates were members of cluster II (moderately slow growth). Different PvuII-RFLP patterns were detected in different sheep flocks from Morocco and South Africa. Our results demonstrate that genetically and phenotypically different strains of M. paratuberculosis were present in ruminant populations. The strains from sheep in Morocco and South Africa tested in the study appeared to belong to a unique group of M. paratuberculosis strains that might have adapted to this host species. The presence of several genetically distinct strains in different sheep flocks suggested that analysis of IS900-specific RFLP patterns may provide a useful tool for the epidemiologic investigation of ovine paratuberculosis outbreaks.

Summary Fecal Escherichia (E.) coli strains from calves were tested for simply detectable phenotypical features associated with Shiga-like toxin (SLT) genes. DNA hybridization with SLT -specific oligonucleotide gene probes (detection of genes for SLT-I and SLT-II ) was the “gold standard” for the evaluation of Vero cell cytotoxicity, fermentation of several saccharides, β-D-glucuronidase activity and production of α-hemolysin α-Hly) or enterohemolysin (EHly). While SLTEC and non- SLTEC did not significantly differ in production of α-Hly, β-D-glucuronidase activity and fermentation of D-sorbitol, production of E-Hly and non-fermentation of L-rhamnose (Rha) and D-sucrose (Suc) were associated with SLT genes. Sensitivity and specificity of the E-Hly + phenotype were 53 % and 8 8 % for identification of calf SLTEC . When three markers were combined to form the parameter E-Hly + or (Rha − and Suc − )”], sensitivity was higher (65%) and specificity was almost the same (85%). Production of enterohemolysin and inability to ferment rhamnose and sucrose were more often associated with the SLT-I gene than with SLT-II genes. Approximately 71% SLT-I + E. coli were positive in the enterohemolysin assay. The test combination “E-Hly + or (Rha − and Suc − )” was most valuable for the presumptive identification of SLT-I + E. coli (sensitivity 85%, specificity 83%). These data suggest that the phenotype “E-Hly + or (Rha − and Suc − )” may be a helpful marker for the detection of SLT-I + E. coli in SLTEC associated diarrhoea of calves.

The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene ( plc ) by colony blot hybridization assay. A vector free digoxigenin—dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C. perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C. perfringens were tested for plc . None of the strains failed in hybridization. Presence of plc was even demonstrated in C. perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp. tested, e.g. C. bifermentans, C. tertium, C. novyi, C. chauvoei, C. sporogenes, C. difficile, C. putrificum, C. sordellii, C. botulinum, C. septicum and C. histolyticum , hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C. perfringens plc .

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.

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.

Aims: Microbiological findings in harbour porpoises from different regions of the North Atlantic were compared. Results in animals from the North and Baltic Seas were evaluated over a period of 18 years for changes in the microbiological flora. Methods and Results: Microbiological investigations were performed on 1429 organ samples from the lung, liver, kidney, spleen, intestine, and mesenteric lymph nodes from harbour porpoises of the German North and Baltic Seas, Greenlandic, Icelandic and Norwegian waters. A large variety of bacteria, including potentially pathogenic bacteria like Brucella sp., Clostridium perfringens, Escherichia coli, Erysipelothrix rhusiopathiae, β-haemolytic streptococci and Staphylococcus aureus were isolated. Those bacteria were associated with bronchopneumonia, gastroenteritis, hepatitis, pyelonephritis, myocarditis and septicemia. Conclusions: Organs from animals originating from Greenlandic and Icelandic waters showed clearly less bacterial growth and fewer associated pathological lesions compared to animals from the German North and Baltic Seas and Norwegian waters. Significance and Impact of the Study: Differences in bacterial findings and associated lesions between harbour porpoises from the German North and Baltic Seas and animals from Greenlandic, Norwegian and Icelandic waters may result from higher stress due to anthropogenic activities such as chemical pollutants in the North and Baltic Seas.

In this study, organ samples from 426 common seals Phoca vitulina, 298 harbour porpoises Phocoena phocoena, 34 grey seals Halichoerus grypus and 10 other marine mammals were assessed for the presence of Brucella species. Forty-seven common seals, 2 harbour porpoises and 1 grey seal were found to be positive for these bacteria. A total of 91 Brucella strains were successfully isolated, due to the fact that Brucella spp. were found in more than one organ sample in 15 animals. The primary organ in which the bacteria were present was the lung. In addition, 2 strains were isolated from lungworms (Parafilaroides spp.). Forty-nine of the isolated strains were selected for further analysis using conventional phenotyping methods. Molecular characterisation was carried out by analysing the IS711 and omp2 loci. With respect to the distribution of the IS711 loci in the genome, the 49 field isolates differed strongly from the terrestrial Brucella species and marginally from the marine Brucella reference strain NCTC12890. Based on the results of the PCR restriction fragment length polymorphism (PCR-RFLP) investigation of the omp2 locus, the majority of the Brucella field isolates were classified as B. pinnipediae, recently proposed B. pinnipedialis, possessing 1 omp2a gene and 1 omp2b gene. Two field isolates revealed the presence of 2 omp2a genes, as has been described for Brucella ovis. To our knowledge, these results confirm for the first time the presence of Brucella species in the marine mammal population of the German North Sea. These findings highlight the need for additional research on the relevance of these Brucella species for marine hosts and their environment.

In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.

Resistance to cephalosporins and/or fluoroquinolones by Staphylococcus intermedius has remained low in Europe, with effective drugs generally available for systemic therapy in pets. However, multiresistant, mecA-positive S. intermedius isolated from dogs and cats is now emerging in Europe. Twelve S. intermedius isolates, highly resistant to at least five antimicrobial classes, were isolated from skin and ear infections in 11 dogs and a cat. The 12 isolates represented 23% of all S. intermedius submissions from one veterinary dermatology referral clinic in northern Germany to veterinary diagnostic laboratories during an 18-month period and resistance included cefalexin, methicillin and enrofloxacin. The animals had been referred to the clinic with recurrent superficial pyoderma, deep pyoderma, pododermatitis or chronic otitis, all unresponsive to systemic beta-lactam-antibiotics or fluoroquinolones. Infection resolved in 10 dogs and the cat on a combination of antimicrobial treatment and correction of underlying causes. Four dogs and a cat required systemic and topical therapy; in six dogs topical antimicrobial therapy alone was successful. Phenotypic and genotypic characteristics of the S. intermedius isolates were determined; species identification was confirmed by polymerase chain detection of thermonuclease genes (nuc) and the presence and expression of the gene conferring resistance to all beta-lactam antibiotics (mecA) were demonstrated in all; based on pulsed-field gel electrophoresis, six were indistinguishable, the others closely or possibly related. The emergence of multiresistant, mecA-positive S. intermedius in Europe is alarming. Zoonotic implications, awareness among veterinary laboratories and strategies for the use of antimicrobials in small animal practice need to be considered.

Summary In the present study, previously characterized Staphylococcus hyicus isolated in Russia (n = 23) and Germany (n = 17) were investigated for the prevalence of the exfoliative toxin encoding genes exhA, exhB, exhC and exhD by multiplex PCR resulting in the detection of exhD positive strains among the S. hyicus isolated from pigs with exudative epidermitis in Russia and the detection of exhC and exhD for one and two strains isolated from exudative epidermitis in Germany respectively. The toxin gene negative strains were generally isolated from apparently healthy pigs, from other animals and from specimens where the relation between the isolation of S. hyicus and the clinical symptoms remained unclear. Partial sequencing of the toxin genes of selected exhC and exhD positive strains and comparing the sequencing results with sequences of exhC and exhD reference strains revealed an almost complete identity. The results of the present study were in agreement with the findings of Andresen and Ahrens (J. Appl. Microbiol., 96, 2004, 1265) and Andresen (J. Vet. Rec., 157, 2005, 376) that the presented multiplex PCR could be used to investigate S. hyicus for toxinogenic potential and that there is an association between the presence of toxin genes in S. hyicus strains from exudative epidermitis. However, comparable with the S. hyicus strains isolated in Germany which were investigated previously by Andresen (J. Vet. Rec., 157, 2005, 376), exhD seems to predominate in S. hyicus strains from Russia.

The present study was designed to identify and compare 32 beta-hemolytic streptococci isolated from 28 different harbor seals of the German North Sea during the phocine distemper outbreak in 2002. The bacteria were identified as Streptococcus equi subsp. zooepidemicus based on cultural, biochemical, serological and molecular studies. Epidemiological investigations by PCR restriction fragment length polymorphism analysis of the 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 32 strains appeared to be identical. These results indicate that a single bacterial clone seemed to be distributed among the harbor seal population of the German North Sea during this outbreak.

A study of 37 by-caught harbour porpoises from Icelandic and Norwegian waters showed that most were in good or moderate nutritional condition and none was severely emaciated. Mild infection with lungworms (Halocercus invaginatus, Pseudalius inflexus, Torynurus convolutus) was found in 84% of the Icelandic and 91% of the Norwegian animals, usually associated with bronchopneumonia which was rarely severe. Most (91%) of the animals had parasites in the stomach and intestine (Anisakis simplex, Contracaecum osculatum, Pholeter gastrophilus), and Campula oblonga was present in the liver and pancreas of 88 and 21%, respectively. Oesophagitis, gastritis, cholangitis, pericholangitis, pancreatitis and lymphadenitis were almost exclusively associated with parasitic infection and usually mild. Bacterial isolates were obtained from 50 to 55% of the animals but were not considered to be clinically significant. There was no indication of morbillivirus infection. Icelandic and Norwegian animals showed a thicker blubber layer and a lower incidence of severe lesions, especially in the respiratory tract, as compared with reports of by-caught animals from the Baltic Sea.

We investigated the virulence properties and clonal relationship of 21 Escherichia coli strains of serogroup O78 isolated from diarrhoeic cattle and calves. Isolates were screened for 18 genes representing virulence features of different Escherichia coli pathotypes. None of the strains harboured enterotoxin-genes estIa/Ib, eltIa/Ib, or Shiga toxin (stx) genes, genes involved in adhesion (eae, f5, f41) hemolysin gene hlyA or invasion gene ipaC. With a high prevalence we detected enterotoxin astA (61.9%), genes involved in iron acquisition, like fyuA, irp (each 57.1%) and iucD (81.0%), and the operon sequence of Colicin V plasmids (38.1%). Some strains possessed toxin genes cdt-IIIB and cnf1/2 (both 14.3%), the invasion gene tia (23.8%), and the serine protease encoding gene espP (23.8%). Moreover, we could show that E. coli O78 strains under investigation were able to adhere to and invade MDBK-cells with varying efficiencies. The results indicate that the closely related O78 strains, constituting two major PFGE-clusters, harbor various virulence features for bovine intestinal disease but cannot be grouped into one of the common E. coli intestinal pathogenic or other pathotypes according to their virulence gene pattern. Nevertheless, the ability to adhere, invade or harbor toxin genes lets us suggest that O78 strains isolated from diarrheal cases in bovines urges further investigations on the zoonotic potential of these strains.

In the present study streptococci of serological group B isolated from canines (n=48) and felines (n=7) were comparatively investigated with group B streptococci from humans and bovines for cultural, biochemical and serological properties for antibiotic resistancies and by molecular analysis. An identification was performed with group B-specific antiserum, biochemical reactions, by PCR amplification and subsequent endonuclease digestion of the 16S rRNA gene and by amplification of species-specific parts of the 16S rDNA the 16S-23S rDNA intergenic spacer region and the CAMP factor gene cfb. Phenotypic similarities of group B streptococci of canine and feline origin with group B streptococci from humans and differences to group B streptococci of bovine origin could be observed in lactose fermentation, serotype patterns, pigmentation, growth properties of the bacteria in fluid medium and soft agar, hemagglutination reactions and in minocycline and tetracycline resistance. A negative hyaluronidase plate test, a hylB amplicon with a size of 4.6 kb and an insertion sequence 1548 could be observed among canine, feline and human group B streptococci of serotype III. The remaining hyaluronidase positive strains, also including all isolates of bovine origin, had a hylB gene with a size of 3.3 kb. Further genotypic differences could be observed in the occurrence of the genes lmb and scpB which appeared generally among canine, feline and human group B streptococci, but less pronounced among bovine isolates of this species. According to the presented data group B streptococci of canine and feline origin seemed to be more related to human than to bovine isolates of this species possibly indicating some epidemiological relation.

The nucleotide sequence encoding the Salmonella plasmid virulence factor D (SpvD) was determined in 17 Salmonella strains that were different in O and H antigen patterns, animal host and geographical origin, and year of isolation. Nucleotide sequence comparison revealed the existence of at least nine spvD alleles resulting in 8 SpvD protein variants although the nucleotide sequences were highly similar (identity 98.8-100%). The spvD gene products differed from each other in up to 4 amino acid residues only with the exception of the carboxy-terminally truncated SpvD variant of one S. Gallinarum field isolate. The highly conserved primary structure of SpvD in epidemiologically relevant salmonellae suggests that this virulence factor is a promising antigen candidate for diagnostic purposes (i.e. antibody detection in infected animals) but also for immunoprophylaxis in farm animal species.

This study was designed to identify and characterize further Streptococcus agalactiae isolated during routine diagnostics from three diseased dogs and a cat, as well as from the inner organs of a monkey which died on a sepsis with beta-haemolytic streptococci. The cultures could be identified as streptococci of serological group B by cultural, biochemical and serological properties and by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S ribosomal DNA. A further characterization of the isolates by serotyping and by determination of antibiotic resistances revealed a close relationship of these isolates to the human biotype of this species.

Streptococcus porcinus reference strains and routine isolates belonging to Lancefield's serogroup E, P, U and V and to various serotypes of serogroup E were examined for their 16S ribosomal DNA fingerprint pattern. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify gene fragments by polymerase chain reaction from genomic DNA. The amplified 1450 bp fragment was subsequently digested with the restriction enzyme BpiI resulting in two fragments with a size of approximately 1250 by and 200 bp. All 45 S porcinus investigated in the present study could be identified on the basis of this characteristic 16S rDNA fingerprint pattern and clearly differentiated from 16 control strains of various species and serogroups of genus Streptococcus. The present results demonstrate the potential application of 16S rDNA analysis for identification of S porcinus, a species which might express various group- and type-specific antigens.

Summary A nested PCR (nested flagellin PCR) carrying an internal E. coli DNA control was established and compared with an in-vitro culture method for the detection of Borrelia burgdorferi in urine specimens of dogs. The predicted specific amplicon of the flagellin gene fla was generated from all cultured strains of B. burgdorferi tested (comprising three European genospecies). In contrast, all 13 strains of seven other flagellated bacterial species were negative. The PCR detection limit yielded 20 cells of B. burgdorferi per ml of double-distilled water and approx. 250 bacteria per ml of dog urine. Using the bacterial culture method, urine specimens collected from 216 dogs in Germany were all diagnosed negative for spirochetes by in-vitro culture and dark-field microscopy. In contrast, DNA of B. burgdorferi was detected in 32 specimens (14.8%) by PCR. 31 urine specimens (14.4%) showed inhibitory activity in the PCR assay. However, 94 (44%) were inhibitory in the culture assay. The majority of the PCR-positive dogs exhibited major clinical symptoms which have not been reported in the course of B. burgdorferi infection previously, e.g. cystitis (14/32 dogs) or prostatitis (5/32 dogs). Our results indicate that the analysis of urine specimens by the nested flagellin PCR is a highly valuable procedure for the diagnosis of B. burgdorferi infections in dogs.

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.

Post-mortem examinations were performed on two marine toads, one animal showing neurological disorders and the other multifocal dermatitis. In one case, lesions consisted of a severe granulomatous encephalomyelitis and in the other of multiple granulomas in the nasal cavity, lungs, heart, bone marrow, ovaries and skin. Histologically, the lesions revealed varying amounts of dark brown fungal elements, predominantly sclerotic bodies indicative of a mycotic infection due to a pigmented fungus.

Summary For the determination of a species-specific antigen of Streptococcus (S.) equi, acid extracts of group C streptococcal strains from horses (S. equi, S. zooepidemicus, S. equisimilis) were investigated using polyacrylamide gel electrophoresis and the immunoblotting technique. Using sera of horses suffering from strangles as well as sera from horses with respiratory infection of unknown etiology, Western blotting yielded more or less multiple banding reactions with bands in the 70, 54, 42, 40, and 31–28 kd molecular weight ranges against extracts of all of the 3 different bacterial species. However, an antigen found in this study at 18–16 kd which was highly sensitive to trypsin, proved to react specifically and regularly only with the serum of horses exposed to S. equi. The specificity of the reaction was assured by antisera of rabbits and horses vaccinated against S. equi, S. zooepidemicus or S. equisimilis, respectively, and by cross absorption of a serum originating from a mare recovered from strangles with Lancefield group C and group G streptococci as well as a strain of S. pyogenes. According to Western blot results on 180 serum pairs from horses with clinical signs of respiratory infection, 15.6% of which gave positive reactions, the 18–16 kd antigen appears as a marker suitable for qualitative testing of horse sera for antibodies to S. equi.

Summary The aim of the present study was to investigate porcine reference and field strains of the species Haemophilus (H.) pleuropneumoniae and H. parasuis, as well as H. Taxon “minor group” and Taxon C on their amino acid and carbohydrate metabolism by thin layer chromatography. The 17 reference strains studied showed almost identical results within the different species and taxa in both, amino acid and carbohydrate metabolism patterns. Based on a few differing enzymatic reactions a reduced species-and taxon-specific reaction pattern could be established, which included L-alanin, L-citrullin and L-threonin of the amino acids as well as D-ribose, α-D-xylose, mannitol, trehalose, β-melibiose and α-lactose of the carbohydrates. This differentiation system allowed a reliable identification of 7 field strains whereas 4 additional ones, hitherto pre-classified as H. parasuis, could not be associated with any of the above species and taxa.

Summary A total of 110 strains of β-hemolytic streptococci, belonging to serogroup C ( Lancefield ), isolated from horses (71 S. zooepidemicus , 27 S. equisimilis and 12 S. equi ) as well as 5 reference strains were tested for their ability to produce hyaluronidase. The determinations were carried out in a culture test on agarose gel and in a liquid test system (turbidity test according to DiFerrante ). The results of both methods used showed that the three Streptococcus species could be differentiated by the relative quantitative determination of hyaluronidase activity. S. equisimilis strains produce 5 to 10 times more hyaluronidase than those of S. zooepidemicus . The strains of S. equi did not show enzyme production. In addition parallel tests by determination of final N-acetyl-D-glucosamine groups and by turbidity test were done to confirm the hyaluronidase activity of S. equisimilis strains. The comparison of results (Fig. 3) obtained from these tests, showed a good correspondence as demonstrated by a correlation index of 0,93.

Summary On some problems in the demonstration of Haemophilus somnus-antibodies Agglutination titres against Haemophilus (H.) somnus were found in 226 (67.1%) of 337 serum samples of cattle collected at random with titres ranging between 1:8 and 1:128. Comparative testing of H. somnus-antigens prepared from 25 field isolates and 2 references strains against a rabbit-antiserum revealed titre differences from 1:20 to 1:320, suggesting antigenic varieties of the strains. Using an enzyme-immuno-assay similar differences could not be detected in 8 H. somnus-strains, even when the antigens had been extracted by heat, guanidinium chloride, sodium dodecylsulfate or phenol, respectively. Comparative investigations with antigenic preparations of other gram-negative bacterial species (H. parasuis, H. pleuropneumoniae, H. equigenitalis, Pasteurella haemolytica) resulted in a significant cross reactivity of the various antigens. A better differentiation against the heterologous species could be obtained by phenol-extracted antigens of H. somnus implicating that those antigen-preparations, if further purified, may be useful for application in enzyme-immuno-test-systems. Zusammenfassung Bei serologischen Untersuchungen von 337 willkurlich ausgesuchten Rinderseren auf agglutinierende Antikorper gegen Haemophilus (H.) somnus wurden in 67,1% der Seren Agglutinationstiter zwischen 1:8 und 1:128 festgestellt. Das verwendete Antigen wurde aufgrund einer Vergleichsuntersuchung von Antigenpraparationen aus 25 verschiedenen Feldisolaten und 2 Referenzstammen an einem Kaninchen-Immunserum ausgewahlt. Dabei waren bei einzelnen Antigenen Unterschiede in der Hohe der Agglutinationstiter zwischen 1:20 und 1:320 aufgetreten, was auf Antigen-Variationen zwischen H. somnus-Stammen hindeutet. Derartige Stamm-Unterschiede konnten allerdings im ELISA an dem gleichen Kaninchen-Immunserum nicht beobachtet werden. Auch die Verwendung von H. somnus-Antigenen verschiedener Herstellungsart (Hitze-, Guanidiniumchlorid-, Natriumdodecylsulfat-, Phenol-Behandlung) ergab keine signifikanten Differenzen zwischen 8 untersuchten Stammen. Auserdem zeigte sich im ELISA ein hoher Grad an Kreuzreaktivitat zwischen H. somnus-Antigenen und entsprechenden Praparationen anderer gramnegativer Bakterienarten (H. parasuis, H. pleuropneumoniae, H. equigenitalis, Pasteurella haemolytica). Lediglich ein Phenolextrakt-Uberstand-Antigen von H. somnus wies eine zufriedenstellende Spezifitat auf.

SummaryOver a thirteen year period (1965 to 1977) a total of 4790 skin scrapings and hair samples of animals were examined mycologically. 887 strains of dermatophytes were isolated out of 885 of these samples (=18,5%). Most frequently Trichophyton verrucosum was identified in samples from cattle, followed by Microsporum canis isolated from cats, dogs and zoo animals. T. mentagrophytes was mainly found on guinea pigs, chinchillas and dogs and T. equinum on horses. Although the total number of the samples examined within the last 8 years increased, the total of the dermatophytes isolated remained proportionately the same. The relative numbers of the various species of dermatophytes isolated did not change within the period of investigation.