Decontamination of a BSL3 laboratory by hydrogen peroxide fumigation using three different surrogates for Bacillus anthracis spores.

AIMS: Two independent trials investigated the decontamination of a BSL3 laboratory using vaporous hydrogen peroxide and compared the effect on spores of Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis as surrogates for Bacillus anthracis spores, while spores of Geobacillus stearothermophilus served as control. METHODS AND RESULTS: Carriers containing 1·0 × 10(6) spores were placed at various locations within the laboratory before fumigation with hydrogen peroxide following a previously validated protocol. Afterwards, carriers were monitored by plating out samples on agar and observing enrichment in nutrient medium for up to 14 days. Three months later, the experiment was repeated and results were compared. On 98 of 102 carriers, no viable spores could be detected after decontamination, while the remaining four carriers exhibited growth of CFU only after enrichment for several days. Reduction factors between 4·0 and 6·0 log levels could be reached. CONCLUSIONS: A validated decontamination of a laboratory with hydrogen peroxide represents an effective alternative to fumigation with formaldehyde. Spores of B. cereus seem to be more resistant than those of G. stearothermophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide important results in the field of hydrogen peroxide decontamination when analysing the effect on spores other than those of G. stearothermophilus.

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group.

AIMS: To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores. METHODS AND RESULTS: Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis, e.g. lack of beta-haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis. PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis, but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an AluI restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different. CONCLUSIONS: Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.

[Efficient killing of anthrax spores using aqueous and alcoholic peracetic acid solutions]. / Effiziente Abtötung von Milzbrandsporen durch wässrige und alkoholische Peressigsäure-Lösungen.

We analysed the sporicidal effect of different concentrations of aqueous and alcoholic peracetic acid (PAA) solutions on anthrax spores in suspension and germ carrier tests. In activation of anthrax spores in suspension assays was achieved in less than 2 min using 1% PAA solution and in less than 3 min using 0.5% PAA solution, respectively. In contrast, in germ carrier as says, a test under practical conditions, spores on 38% of the germ carriers survived treatment with 1% PAA solution for 15 min. The use of PAA in 80% ethyl alcohol outclassed the sporicidal effect of aqueous PAA solutions in both suspension and germ carrier assays. Anthrax spores on 14% of germ carriers tested survived 30 min of treatment with a 1% aqueous PAA solution. In contrast anthrax spores were reliably inactivated under the same test procedure using a 1% alcoholic PAA solution for 30 min. The proven enhancement of the sporicidal effect of alcoholic PAA solutions should be kept in mind when using disinfectants in practice. In further surveys we will optimise the test conditions.

[The relationship of plasmids from environmental Yersinia isolates and the virulence plasmid of enteropathogenic Yersinia strains]. / Untersuchungen zur Verwandtschaft der Plasmide aus Yersinia-Umweltisolaten und dem Virulenzplasmid enteropathogener Yersinien.

The human pathogenic strains of Yersinia harbour a conserved plasmid carrying the Yop virulon. The virulence plasmid of Yersinia enterocolitica strains belonging to the serogroups O:3 and O:9 were used as probes to detect homologous sequences in plasmids of "avirulent" Yersinia strains. "Avirulent" Yersinia strains (Y. enterocolitica biogroup 1A, Y. intermedia, Y. kristensenii and Y. frederiksenii) lack the virulence plasmid. They are widely distributed in the environment and can frequently be isolated from clinical samples. Hybridisation experiments revealed a number of common genetic elements of the virulence plasmid and the plasmids of "avirulent" Yersinia strains. These elements were identified as genes involved in plasmid replication, as an endonuclease gene and as mobile genetic elements. However, none of the plasmid encoded virulence genes was present in the plasmids of "avirulent" Yersinia strains. The frequent occurrence and the possible etiological relevance of "avirulent" isolates will be discussed.

Evaluation of a systemic antibiotic treatment of toxic puerperal metritis in dairy cows.

The objective of this study was to evaluate the efficacy and economic efficiency of a systemic treatment of toxic puerperal metritis in dairy cows with ceftiofur. Cows with abnormal vaginal discharge at a postpartum examination (d 4 to 6 after calving) and a rectal temperature > or = 39.5 degrees C were assigned to three treatment groups. Cows in group 1 (n = 70) received 600 mg of ceftiofur intramuscularly (i.m.) on 3 consecutive days. Cows in group 2 (n = 79) received an intrauterine treatment with antibiotic pills consisting of 2500 mg of ampicillin and 2500 mg of cloxacillin and an additional 6000 mg (i.m.) of ampicillin. This treatment was performed on 3 consecutive days. Cows in group 3 (n = 78) received the same intrauterine treatment as in group 2. In addition, 600 mg of ceftiofur was administered i.m. on 3 consecutive days. Body temperature was recorded daily for 6 d after first treatment. There were no significant differences among the groups regarding clinical efficacy at d 6 after first treatment. The cure rates based on rectal temperatures declining to below 39.5 degrees C on d 6 after treatment were 82.9, 84.8, and 84.6% for groups 1, 2, and 3, respectively. Reproductive performance did not differ significantly between group 1 and groups 2 and 3 for any of the measures tested. A financial analysis with 87 different cost scenarios demonstrated that a systemic treatment of toxic puerperal metritis in cattle with ceftiofur is an effective alternative to the combination of local and systemic treatments.

Treponema brennaborense sp. nov., a novel spirochaete isolated from a dairy cow suffering from digital dermatitis.

A novel Treponema species was isolated from an ulcerative lesion of a cow suffering from digital dermatitis (DD), a disease which causes painful ulcerations along the coronary band. Among other anaerobic bacteria, high numbers of spirochaetes have been regularly found in DD lesions. Here data are presented of a spirochaete isolated from a DD ulcer. By chemotaxonomy, protein analysis and comparative 16S rDNA sequence analysis this isolate was classified as a treponeme that differed from all Treponema species described previously. The only isolate, DD5/3T, for which the name Treponema brennaborense is proposed, is designated the type strain of the novel species. The strain is a small, highly motile spirochaete that has two periplasmic flagella, one flagellum being attached at each cell pole. Strain DD5/3T exhibits alpha-glucosidase and N-acetyl-beta-glucosaminidase activity and growth is inhibited by rabbit serum. T. brennaborense was phylogenetically most closely related (89.5% 16S rRNA similarity) to Treponema maltophilum, an oral spirochaete isolated from a periodontitis patient.

Characterization of plasmid regions of foodborne Yersinia enterocolitica biogroup 1A strains hybridizing to the Yersinia enterocolitica virulence plasmid.

The aim of our study was to find out if plasmids of foodborne Yersinia enterocolitica biogroup 1A strains harbour genes related to the virulence genes located on the virulence plasmid pYV of Yersinia enterocolitica. The foodborne strains were isolated from pork, as pigs are considered as an important reservoir for enteropathogenic Y. enterocolitica 0:3 and 0:9 strains. The plasmids of the foodborne strains were characterized by restriction enzyme analysis and hybridized to the virulence plasmid pYV of pathogenic Y. enterocolitica strains (0:3 biogroup 4; 0:9 biogroup 2). In several cases the plasmids of the foodborne strains showed homologies to parts of the pYV plasmid. Analysis of the hybridizing regions revealed that genes involved in replication, sequences of transposable elements and an endonuclease gene caused the observed hybridization to the virulence plasmid. In cause of the study also a remnant of a Tn3-like transposon was shown to be present adjacent to the yadA gene on the pYV plasmid. Although there is evidence that at least some strains of Y. enterocolitica biogroup 1A might possess pathogenic properties none of the well known plasmid encoded virulence genes were present on the plasmids of the investigated foodborne biogroup 1A strains.

Spirochetes from digital dermatitis lesions in cattle are closely related to treponemes associated with human periodontitis.

Digital dermatitis (DD), first described in 1974 by Cheli and Mortellaro (R. Cheli and C. Mortellaro, p. 208-213, in Proceedings of the 8th International Conference on Diseases of Cattle, 1974), is a major problem in diary cows and beef cattle causing significant economic losses worldwide. Lesions are typically found at the volar skin proximal to the heel bulbs. Microscopic examination of biopsies or touch preparations of these lesions revealed a variety of different bacterial morphotypes including significant numbers of spirochetes which often represent the predominant morphotype. We used comparative 16S rRNA sequence analysis to determine the diversity and phylogeny of these hitherto unclassified DD spirochetes. Results indicate that those lesions looked at so far contained at least five spirochetal phylotypes, all clustering within the genus Treponema. Phylotype DDKL-4 was nearly identical (99.4% similarity) to that of a nonpathogenic human treponeme, T. phagedenis. Two phylotypes DDKL-3 and DDKL-13 were closely related to those from treponemes commonly found in human periodontitis lesions, i.e., T. denticola and T. vincetii, exhibiting 95 and 98% similarity, respectively. The other two phylotypes, DDKL-12 and DDKL-20, had no close relatives to any cultivable treponemal species but clustered to previously described group IV oral treponemes. Preliminary analysis using in situ hybridization with fluorescently labeled oligonucleotide probes against smears from DD biopsies revealed that from all lesions analyzed so far, T. denticola-like spirochetes were detected in the highest proportion of all spirochetal morphotypes.

Comparison of plasmids of strains of Yersinia enterocolitica biovar 1A with the virulence plasmid of a pathogenic Y. enterocolitica strain.

The plasmid content of apathogenic Y. enterocolitica biovar 1A strains was determined and the plasmids were compared with the virulence plasmid of a pathogenic Y. enterocolitica strain. About 38% of the selected biovar 1A strains contained plasmids of different sizes ranging from 2.7 kb to more than 70 kb. Some of the larger plasmids had a size similar to that of the virulence plasmid of a pathogenic reference strain. The restriction patterns of these plasmids were different from the restriction pattern of the virulence plasmid of the pathogenic reference strain. Differences were also observed in hybridization studies with the virulence plasmid. The plasmids from 15 out of 16 biovar 1A strains showed no homology, whereas the plasmid of one biovar 1A strain partially hybridized to the virulence plasmid.

[Electron microscopic detection of spirochetes in dermatitis digitalis of cattle]. / Zum elektronenmikroskopischen Spirochäten-Nachweis bei der Dermatitis digitalis des Rindes.

In typical Dermatitis digitalis (D.d.) lesions, spirochaete-like bacteria with variations in spiralization were revealed by electron microscopy. While, in the early stages of the disease, these are found to be associated with fibrillar material of keratocytes, they occur massively in vacuoles at more advanced stages. The spirochaetes carry one pair of endoflagella, originating with a hook from the poles of the bacteria. These flagellae are composed of coiled flagellating fibrils in the pole region, merging towards the centre of the bacterium. A coat of fibrils was found in association with the cytoplasmatic membrane. The winding of this coat follows and may influence the coiling of the protoplast, and is probably involved in the rapid motility of this spirochaetes, together with the flagella. Immuno-electronmicroscopy revealed an antigenic relationship with Borrelia burgdorferi, at least with regard to the regions of flagella and undulating membrane. The paper discusses: 1. The possible classification of these spirochaetes with the genus Treponema; 2. The layer of peptidoglycan occurring on the outer membrane; and 3. The keratolytic activity of spirochaetes in D.d.

[Immunostimulating effect of culture filtrates of Yersinia enterocolitica]. / Immunstimulierende Wirkung durch Kulturfiltrate von Yersinia enterocolitica.

Application of sterile culturing supernatant of Yersinia (Y.) enterocolitica (tested serovars were 03 and 08) caused significantly accelerated transplant rejection in mice of various inbreeding strains. Action correlated with dosage (r = -0.92). C57B16 mice were tested for their pregnancy rates in response to the same filtrate (serovar 03), with 5.5 live births per mating being recorded from 47 control matings but only 4.4 from 45 experimental matings (alpha less than 0.0025). The mean gestation period of experimental animals was extended by five percent over that of simultaneous controls (alpha = 0.25). Particular reference is made in this paper to Vesikari et al. (1987) who found Y. enterocolitica to function as interleukin-1 inductor via lipopolysaccharide. The active substance tested in this context, however, proved to be thermolabile, with 30 minutes of heating to 56 degrees C eliminating the action tested before. Y. enterocolitica infections were frequently found to coincide with rheumatoid arthritis, and evidence has been produced to the unspecific stimulating effect of Y. enterocolitica culture filtrates (testing being applied to serovar 03, biovar 4 and serovar 08, biovar 2). It is against the background of these aspects that chronic and other infections by Y. enterocolitica are considered to be of substantive relevance to the etiopathogenesis of autoimmune diseases, above all rheumatoid arthritis.

[Brucella clearance as a sensitive method for the detection of cross reactions of Brucella abortus with Yersinia enterocolitica 03, 06, 09 and Salmonella urbana and Salmonella abony]. / Brucella-Clearance als sensitive Methode zum Nachweis der Kreuzreaktionen von Brucella abortus mit Yersinia enterocolitica O3, O6, O9 sowie Salmonella urbana und Salmonella abony.

Determination of the Brucella clearance rate has proved to enable assessment of Brucella immune reaction in rat, even after vaccination with Yersiniae and Salmonellae. Vaccination with Yersinia (Y.) enterocolitica O6 and O9 produced 95 per cent of "high responders", whereas 65 per cent of "high responders" and 25 per cent of "non-responders" were recorded in the wake of O3. Salmonella (S.) urbana vaccination gave 50 per cent of "high responders" and 27 per cent of "non-responders", while 100 per cent "non-responders" resulted from S. dublin. Vaccination, using Brucella abortus Buck 19, gave 100 per cent "high responders". The differentiated nature of immune reactions to Y. enterocolitica O3, S. urbana, and S. abony has been attributed to an individual genetic capability of reaction to the cross-reactive antigen.

[Brucella titers in swine caused by Yersinia enterocolitica, serotype 0:9]. / Brucellatiter beim Schwein, hervorgerufen durch Yersinia enterocolitica Serotyp 0:9.

Antibody to Brucella abortus developed in two thirds of all gilts kept on a pig breeding station. Systematic tests taken for the purpose of detecting clinical symptoms and of isolating Brucella were negative. however, Yersinia (Y.) enterocolitica, Serotype 0:9, was cultured from rectal swab samples which had been obtained from 31 to 78 gilts. The clinical, bacteriological, and serological tests gave rise to the assumption that the Brucella titres have been caused by Yersinia enterocolitica infection. Such conclusion, however, could be drawn only as a result of a complex investigation. Detection of Yersinia enterocolitica alone is not sufficient a proof by which to rule out the possibility of concomitant Brucella infection. The question is discussed to what extent swine may be considered to be a potential source of infection of man.

[Reproduction of Brucella titers in swine using experimental infection with Yersinia enterocolitica, serotypes 0:9 and 0:6]. / Reproduktion von Brucellatitern beim Schwein mittels experimenteller Infektion mit den Yersinia-enterocolitica-Serotypen 0:9 und 0:6.

Measurable Brucella titres were produced by slow agglutination, slow agglutination at 57 degrees C (heat test), and complement fixation reaction with Brucella antigen in infection experiments with gilts in which Yersinia enterocolitica Serotypes 0:9 and 0:6 were used. Slow agglutination gave brucellosis titres up to 1:1280 and titres against Yersinia enterocolitica, Serotype 0:9, up to 1:20480. The antibody titres stayed persistent throughout the 80 days of the experiment. Yersinia enterocolitica infection was found to be transmissible between the animals. Aspects relating to the development and course of the infection as well as to pathogen detection are discussed.

[Staphylococcus aureus infection in chickens in industrialized poultry units. 2. Microbiological studies: Staphylococcus aureus and other pathogens]. / Untersuchungen zur Staphylococcus-aureus-Infektion des Huhnes in Anlagen der industriemässigen Geflügelproduktion. 2. Mitteilung: Mikrobiologische Untersuchungen: Staphylococcus aureus und andere Erreger.

Checks were applied to 3,213 dead or ill broiler chickens and broiler parents for the purpose of elucidating enzootic or epizootic Staphylococcus aureus infections which had occurred on three industrialised poultry units. Rates of Staphylococcus aureus detection and identification declined by the following order: staphylococcal septicaemia (100 per cent), dermatitis (75.42 per cent), arthritis and tenosynovitis or osteomyelitis (64.59 per cent), wound infections (24.02 per cent), infection of bursa suprasternalis (18.94 per cent), underdevelopment (8.70 per cent), navel-yolksac-peritonitis (7.83 per cent), and conjunctivitis (7.14 per cent). Staphylococcus aureus was found to be the most important pathogen of arthritis with synovitis and of osteomyelitis with epiphysiolysis, the rate of detection being directly correlated with the severity of the pathologico-anatomic alterations established. Attempts to culture mycoplasmas from irritated synovial sheaths were successful only in three of 56 examined animals (5.3 per cent). The site variety of gallinae or strains of the crystal-violet Type A with lysis patterns of 84, 53, 77 and 84 accounted for 78.1 per cent of all Staphylococcus aureus strains isolated from infected fowl. Their percentual importance was 85.0 for septicaemia, dermatitis, and arthritis with synovitis and osteomyelitis. For prophylaxis and control of Staphylococcus aureus infections of fowl on elimination of predisposing factors and on the control of epidemic occurrence of the above types.

[The Corynebacterium pyogenes infection in cattle. 1. Incidence of the pathogen]. / Die Corynebacterium-pyogenes-Infektion des Rindes 1. Mitteilung: Verbreitung des Erregers.

Reported are the incidence of Corynebacterium pyogenes together with different pathological changes as well as the existence of latent Corynebacterium pyogenes infections and their widespread occurrence. Corynebacterium pyogenes was established from 609 in 2,130 samples of pathological processes, accounting for 28.6%. The pathogen was cultivated from various processes, including enlarged tail lymph nodes (61.1%), tail phlegmons (56.3%), abscesses (49.1%), epiphysiolyses (45.2%), liver abscesses (31.8%), panaritia at beginning of fattening (20.5%), aborted foetuses (14.9%), foetal membranes in cases of incarcerated placenta (12.0%), and panaritia on end of fattening (3.4%). The same pathogenic microorganism was recorded from nine per cent of apparently intact heifer udders, before pasturing. Corynebacterium pyogenes was cultivated also from nasal mucous membrane (8.4%) and retropharyngeal lymph nodes (37.2%). The highest detection rate was 71.6%, obtained from the tonsils.

[The Corynebacterium pyogenes infection of cattle. 2. Tenacity of Corynebacterium pyogenes]. / Die Corynebacterium-pyogenes-Infektion des Rindes 2. Mitteilung: Tenazität des Corynebacterium pyogenes.

Some common agents were tested for their effectiveness against Corynebacterium pyogenes. The pathogen proved most susceptable to Wofasteril. All germs were killed within ten minutes by a 0.005% solution. Equally good action was recorded from all the other tested agents as well (lactic acid, Lugol's solution, formalin, cupric sulphate, alcohol, and aethacridine. Other studies were conducted with the view to testing the survival capacity of Corynebacterium pyogenes in different media and storage conditions. The pathogen survived three months in routine media and mastitis secretion at room temperature. Regrowth of 38 in 50 strains took place after nine months of refrigerator storage in slanting blood agar tubes with paraffin plugs. Germs sampled from mastitis secretion and stored in a refrigerator were cultivable even after one year had elapsed. The detectability rate of Corynebacterium pyogenes did not change over months by storage of wound infection material at 12 degrees C below zero. The pathogen remained detectable five days from artificial contamination of cattle skin.