Methods for identification and differentiation of different Shewanella spp. isolates for diagnostic use.

Shewanella spp. are Gram-negative, rod-shaped, motile bacteria that are widely distributed in marine and freshwater environments. The bacteria are present in the physiological microflora of fish from temperate waters and are known as fish spoilage species. From clinically healthy fish and from fish with skin ulcerations, Shewanella spp. is regularly isolated, indicating a possible role as fish pathogen. In this study, 74 isolates of Shewanella spp. were analysed. For species identification, biochemical techniques, 16S rRNA sequencing, MALDI-TOF MS and the Sherlock Microbial Identification System (MIS) based on the composition of fatty acid ethyl esters were compared. The phylogenetic relationship, cytotoxicity in vitro and resistance against antibiotics were tested. The most reliable method for species identification was 16S rRNA sequencing. From diseased fish, clinically healthy fish and the aquatic environment, different Shewanella species were isolated. This indicates that Shewanella spp. is widespread in the aquatic milieu and acts as a secondary pathogen. The virulence of Shewanella spp. is probably not depending on the species but on the isolate itself. Many isolates of Shewanella spp. were showing multiresistances against antibiotic substances, especially in samples derived from retailers and in routine diagnostics, all Shewanella spp. should therefore be tested for resistances against antibiotic agents.

Ultraviolet radiation and nanoparticle induced intracellular free radicals generation measured in human keratinocytes by electron paramagnetic resonance spectroscopy.

BACKGROUND: Several nanoparticle-based formulations used in cosmetics and dermatology are exposed to sunlight once applied to the skin. Therefore, it is important to study possible synergistic effects of nanoparticles and ultraviolet radiation. METHODS: Electron paramagnetic resonance spectroscopy (EPR) was used to detect intracellular free radicals induced by ultraviolet B (UVB) radiation and amorphous silica nanoparticle and to evaluate the influence of nanoparticle surface chemistry on particle cytotoxicity toward HaCaT cells. Uncoated titanium dioxide nanoparticles served as positive control. In addition, particle intracellular uptake, viability, and induction of interleukin-6 were measured. RESULTS: We found that photo-activated titanium dioxide particles induced a significant amount of intracellular free radicals. On the contrary, no intracellular free radicals were generated by the investigated silica nanoparticles in the dark as well as under UVB radiation. However, under UVB exposure, the non-functionalized silica nanoparticles altered the release of IL-6. At the same concentrations, the amino-functionalized silica nanoparticles had no influence on UVB-induced IL-6 release. CONCLUSION: EPR spectroscopy is a useful technique to measure nanoparticle-induced intracellular free radicals. Non-toxic concentrations of silica particles enhanced the toxicity of UVB radiation. This synergistic effect was not mediated by particle-generated free radicals and correlated with particle surface charge and intracellular distribution.

Health status of free-ranging ring-necked pheasant chicks (Phasianus colchicus) in North-Western Germany.

Being a typical ground-breeding bird of the agricultural landscape in Germany, the pheasant has experienced a strong and persistent population decline with a hitherto unexplained cause. Contributing factors to the ongoing negative trend, such as the effects of pesticides, diseases, predation, increase in traffic and reduced fallow periods, are currently being controversially discussed. In the present study, 62 free-ranging pheasant chicks were caught within a two-year period in three federal states of Germany; Lower Saxony, North Rhine-Westphalia and Schleswig-Holstein. The pheasant chicks were divided into three age groups to detect differences in their development and physical constitution. In addition, pathomorphological, parasitological, virological, bacteriological and toxicological investigations were performed. The younger chicks were emaciated, while the older chicks were of moderate to good nutritional status. However, the latter age group was limited to a maximum of three chicks per hen, while the youngest age class comprised up to ten chicks. The majority of chicks suffered from dermatitis of the periocular and caudal region of the head (57-94%) of unknown origin. In addition, intestinal enteritis (100%), pneumonia (26%), hepatitis (24%), perineuritis (6%), tracheitis (24%), muscle degeneration (1%) and myositis (1%) were found. In 78% of the cases, various Mycoplasma spp. were isolated. Mycoplasma gallisepticum (MG) was not detected using an MG-specific PCR. Parasitic infections included Philopteridae (55%), Coccidia (48%), Heterakis/Ascaridia spp. (8%) and Syngamus trachea (13%). A total of 8% of the chicks were Avian metapneumovirus (AMPV) positive using RT-PCR, 16% positive for infectious bronchitis virus (IBV) using RT-PCR, and 2% positive for haemorrhagic enteritis virus (HEV) using PCR. All samples tested for avian encephalomyelitis virus (AEV), infectious bursal disease virus (IBDV) or infectious laryngotracheitis virus (ILTV) were negative. The pool samples of the ten chicks were negative for all acid, alkaline-free and derivative substances, while two out of three samples tested were positive for the herbicide glyphosate. Pheasant chick deaths may often have been triggered by poor nutritional status, probably in association with inflammatory changes in various tissues and organs as well as bacterial and parasitic pathogens. Theses impacts may have played a major role in the decline in pheasant populations.

[3D-X-ray microcomputer tomography and optical coherence tomography as methods for the localization of the blastoderm in the newly laid unincubated chicken egg]. / 3D-Röntgen-Mikrocomputertomographie und Optische Kohärenztomographie als Methoden zur Lagebestimmung des Blastoderms im unbebrüteten Hühnerei.

The routine culling of the male offspring of hybrid layer type chickens is met with increasing public disapproval for both ethical and legal reasons. Until now practice-oriented methods for reliable sex diagnosis prior to hatch could not be developed. Molecular genetical analysis of blastodermic cells can be used for sex determination in unincubated eggs; however, knowledge of the precise localization of the germinal disc is crucial for the extraction of a carefully directed cell biopsy. In principle, 3D-X-ray micro computed tomography (3D-CT) has been proven a suitable method to localize the germinal disk in the unincubated egg without damaging the egg shell. No negative effects on embryogenesis and hatching rate of irradiated hatching eggs were established. The pictorial representation of the germinal disk using optical coherence tomography (OCT) failed in the unopened egg. The egg shell formed an impenetrable barrier for the currently available measuring method which utilized near infrared (NIR) wavelength regions. After opening the egg shell, the germinal disk could be visualized without any difficulties. In conclusion, technical possibilities for localization of the germinal disk in the unincubated egg already exist, but regarding technical parameters, the procedures have to be adapted to the specific purpose.

Investigations on infection status with H5 and H7 avian influenza virus in short-distance and long-distance migrant birds in 2001.

The role of migrating birds as potential vectors for avian influenza virus (AIV) was investigated. We captured 543 migrating passerines during their stopover on the island of Helgoland (North Sea) in spring and autumn 2001. These birds were sampled for avian influenza A viruses (AIV), specifically the subtypes H5 and H7. For virus detection, samples were taken from 1) short-distance migrants, such as chaffinches (Fringilla coelebs; n = 131) and song thrushes (Turdus philomelos; n = 169); and 2) long-distance migrants, such as garden warblers (Sylvia borin; n = 142) and common redstarts (Phoenicurus phoenicurus; n = 101). Virus isolation assays failed to identify AIV. Therefore, regarding the actual low number of samples, we speculate that the tested four species of passerines were not infected by AIV, indicating that the passerine species examined in this study may play only a minor role as potential vectors of AIV.

No detection of avian influenza A viruses of the subtypes H5 and H7 and isolation of lentogenic avian paramyxovirus serotype 1 in passerine birds during stopover in the year 2001 on the island Helgoland (North Sea).

A total of 543 migrating passerines were captured during their stopover on the island of Helgoland (North Sea) in spring and autumn 2001. They were sampled for the detection of avian influenza A viruses (AIV) subtypes H5 and H7, and for avian paramyxoviruses serotype 1 (APMV-1). The goal of the study was to examine the role of migrating birds as potential vectors for these zoonotic viral diseases. For virus detection samples were taken from a) short-distance migrants such as chaffinches (Fringilla coelebs, n = 131) and song trushes (Turdus philomelos, n = 169), and b) long-distance migrants such as garden warbler (Sylvia borin, n = 142) and common redstarts (Phoenicurus phoenicurus, n = 101). Virus detection was done on conjunctival, choanal cleft and cloacal swabs. Embryonated SPF chicken eggs were used to isolate and propagate virus followed by virus identification in a hemagglutination test, hemagglutination inhibition test and in an agar gel diffusion test. In none of the tested samples AIV was detected. Therefore, we conclude that the tested four species of passerines were infected by these pathogens. Six out of 543 birds (1.1 %) were found to carry non-pathogenic and lentogenic strains of APMV-1. This indicates that the passerine species examined in this study may play only a minor role as potential vectors of APMV-1.

Fatty acid patterns of different stages of Oesophagostomum dentatum and Oesophagostomum quadrispinulatum as revealed by gas chromatography.

Total methylated fatty acid patterns of various developmental stages (third-stage larvae (L3), L3 and fourth-stage larvae (L4) cultured in vitro, L4 and female and male adults derived from intestinal contents) of the porcine nodular worms Oesophagostomum dentatum and Oesophagostomum quadrispinulatum and their cultivation medium were analysed by gas chromatography using Microbial Identification computer software. Fatty acids ranging from C-12 to C-20 could be separated. For each stage and species, characteristic patterns were found. The most prevalent fatty acids were C-18. The freshly exsheathed larvae contained the greatest variety of fatty acids (including short-chain fatty acids C-12 to C-15) with approximately equal amounts of fatty acids with odd and even chain lengths, whereas more advanced stages consisted of a lower number of fatty acids with mostly even chain lengths >/=C-16. Intestinal stages contained less odd-numbered fatty acids and less branched fatty acids than others. In contrast to intestinal L4, cultivated L4 had high amounts of C-15:0 and C-17:0. Sheathed L3 contained more C-18 than freshly exsheathed ones, and medium incubated for 7 days in the presence of parasites contained C-13 to C-15 and monounsaturated C-16, but less C-18 and C-20:4 than fresh medium or medium incubated without worms. Based on the evaluation of stage- and species-specific fatty acid patterns random samples could be assigned to the correct stage and species. In a dendrogram based on fatty acid patterns the same stages of the two species formed the closest relationships, and the intestinal stages formed a clade distinct from the cultivated larvae and L3. All stages contained considerable relative amounts of arachidonic acid, the main precursor of eicosanoids. The fixed differences between species and stages indicate genetic regulation of fatty acid patterns, while environmental influences are mirrored by differences between cultivated and intestinal stages. Regulation of fatty acid patterns probably plays a role in worm physiology and host-parasite interaction.

Detection of acid production from carbohydrates by Riemerella anatipestifer and related organisms using the buffered single substrate test.

One hundred and twenty-one Riemerella anatipestifer field strains from wild birds, domesticated poultry and pigs were examined for their ability to produce acid from carbohydrates by using conventional biochemical and buffered single substrate (BSS) test methods. The type strains of the species R. anatipestifer and taxometrically related genera Chryseobacterium and Bergeyella were included in the study. In contrast to 10 indole-positive R. anatipestifer variant strains, only a few of the 111 typical indole-negative R. anatipestifer strains produced acid from dextrin (32%), glucose (17%), maltose (14%) and trehalose (5%) when the conventional test procedure was used. Using the BSS test all the field isolates and the type strain of R. anatipestifer produced acid from one or more carbohydrates, most of them from dextrin (96%), maltose (91%), glucose (87%), mannose (83%), less frequently from fructose (38%) and only in some cases from trehalose (19%). One hundred and six (87%) of the R. anatipestifer strains could be assigned to 8 biovars, based on the diversity of the carbohydrate acidification patterns. The remaining 16 R. anatipestifer isolates gave delayed reactions and displayed 13 different carbohydrate acidification profiles. The Chryseobacterium and Bergeyella type strains also produced acid from more carbohydrates when the BSS test was used. The BSS-carbohydrate acidification pattern of the Chryseobacterium indologenes strain was similar to that of R. anatipestifer biovar 3.

[Pilot study of the occurrence of Chlamydia psittaci infections in commercial turkey flocks in Niedersachsen]. / Pilotstudie über das Vorkommen von Chlamydia psittaci-Infektionen in kommerziellen Putenherden Niedersachsens.

Turkeys from 8 commercial flocks, of which were 6 clinically healthy, were examined by use of enzyme-linked immunosorbent assay systems for genus-specific Chlamydia antigens and also for antibodies against Chlamydia psittaci in 4 flocks. Results of the tests indicate the presence of Chlamydia psittaci infections in all 8 flocks. The percentage of combined cloacal and conjunctival swabs positive for Chlamydia antigen ranged from 20-91% [20-70%] whereas the percentage of antibody-positive serum samples ranged from 81-100%. Our findings suggest, that the prevalence of Chlamydia psittaci infections in clinically healthy turkey flocks is higher than hitherto suspected.

[The effect of magnetic resonance treatment on chicken embryos]. / Untersuchungen zur Auswirkung von Magnet-Resonanz(MR)-Behandlungen auf Hühner-Embryonen.

Nuclear Magnetic Resonance (NMR) is of increasing diagnostic importance especially in human medicine. To evaluate possible side effects of this technology, embryonated chicken eggs were used as a model. Different fields (static magnetic field [1 oder 4 T], variable magnetic field [gradient] or high frequency field) were applied before the beginning and at the fifth day of incubation for different times (18.8, 37.6, 56 or 75.1 min, resp.). According to the criteria embryo-mortality, hatching-rate or vitality of the chickens, influences of the NMR-treatment were not observed.

[Evaluation of different broiler housing conditions on the basis of clinical, pathological, anatomical and laboratory diagnostic criteria--a summary]. / Bewertung verschiedener Haltungsbedingungen von Broilern anhand klinischer, pathologisch-anatomischer und labordiagnosticher Kriterien-Eine zusammenfassende Darstellung.

In a cooperative study, the impact of different broiler housing conditions (see communication BUCHENAUER) on clinical, post-mortem and laboratory diagnostic (microbiological, parasitological, serological, immunological) parameters was investigated in the course of three different fattening period. Clinically, it was observed, that lower stocking densities allowed a relatively higher motility of the birds. On the other hand, necropsy findings, microbiological, parasitological, serological and immunological findings gave no indications that these parameters were influenced statistically significant by either one of the broiler keeping systems.

[Relevance and diagnostics of selected bacterial pathogens of poultry]. / Bedeutung und Diagnostik ausgewählter bakterieller Erreger des Geflügels.

This paper provides an overview of diseases caused by Bordetella avium, Gallibacterium anatis, Ornithobacterium rhinotracheale, Riemerella anatipestifer and Enterococcus cecorum in poultry flocks. These bacterial species are almost exclusively found in birds. Their identification with biochemical methods is described and alternative molecular biological methods are discussed.

[The genetic classification of the Pasteurella pneumotropica complex]. / Untersuchungen zur genetischen Klassifikation des Pasteurella-pneumotropica-Komplexes.

Pasteurella pneumotropica with its biotypes Jawetz and Heyl are the most common bacterial pathogens associated with diseases in rodents. 23 P. pneumotropica biotype Jawetz, biotype Heyl and P. pneumotropica-like rodentia isolates have been investigated phenotypically by characterization of their micromorphology and biochemical fermentation reactions. The taxonomic position within the family Pasteurellaceae has been examined by DNA:DNA hybridisation (optical method). It could be shown that P. pneumotropica biotype Jawetz represents a genus-like cluster containing several species including the V-factor dependent Haemophilus Taxon B and the avian P. pneumotropica-like organism and therefore resembles a new species of the new genus. It is concluded that the biotype Heyl of P. pneumotropica taxonomically remains as a species within the family Pasteurellaceae, however without further relationship to other known genera or genus-like groups.

Exclusion of strain 670/89 as type strain for serovar 20 of Riemerella anatipestifer.

Classical phenotypic characterisation and numeric analysis of whole-cell fatty acid patterns of twenty-one type strains of hitherto reported Riemerella anatipestifer serovars revealed that the type strain of serovar 20 does not belong to the species Riemerella anatipestifer sensu stricto and, therefore, it has to be excluded as a representative Riemerella anatipestifer serotype strain.

[Differentiation of Salmonella gallinarum rough and smooth strains using gas chromatography analysis of their cell-bound fatty acids]. / Differenzierung von Salmonella Gallinarum-Stämmen der Rauh- und Glattform durch gaschromatographische Analyse ihrer zellgebundenen Fettsäuren.

The whole cell fatty acids of 7 field isolates of smooth (S)-type S. Gallinarum strains and 3 isolates of rough (R)-type S. Gallinarum strain 9R were determined by the liquid-gas-chromatography using the Microbial Identification System (MIS, Microbial ID, Inc. [MIDI, Newark, Delaware USA]). The profiles of the bacterial cellular fatty acids of the S- and R-type isolates differ in the number as well as in the quantity of the fatty acids. On the basis of these differences the S-type isolates could be distinguished from the R-type cultures.

Use of an alkaline phosphatase-labelled probe for the detection of Mycoplasma synoviae in chickens.

Short nucleotides directly labelled to alkaline phosphatase (SNAP probes) are an interesting alternative to digoxigenin-labelled probes (DIG probes), because they reduce the number of steps necessary in dot blots for the detection of DNA or amplificate. This study examined the questions whether a SNAP probe might not only save time, but also increase the sensitivity of another PCR-based DNA probe test using a digoxigenin probe. Amplificates obtained by multispecies polymerase chain reaction (PCR), with either purified genomic DNA or DNA extracted from tracheal swabs taken in chicken flocks, were detected by both methods. The results for the clinical specimens were compared to culture. Under stringent conditions, the specificity and sensitivity obtained with the SNAP probe were comparable to the results obtained with the DIG probe. The quantities 10 fg (SNAP probe) and 100 fg (DIG probe) of purified Mycoplasma synoviae DNA were detected after amplification, but more positive clinical specimens were detected with the DIG probe. Under non-stringent conditions sensitivity with purified DNA did no change, but the coloration of the dots improved markedly, and more positive specimens could be detected with the SNAP probe than with the DIG probe, truly positives as confirmed by culture. Because cross-reaction with Mycoplasma gallisepticum and Mycoplasma imitans, two species with DNA that was also recognized by the multispecies primers, occurred under non-stringent conditions, it was concluded that, to take the full advantage of SNAP probes, their use in combination with species-specific primer pairs is recommended. PCR as a method for mycoplasma detection is however, always accompanied with serological and cultural methods. When a M. synoviae mono-infection is likely by serological results, non-stringent dot blot conditions and use of the SNAP probe will ease and improve the detection of mycoplasma.

Isolation and identification of Vibrio metschnicovii from domestic ducks and geese.

Seven Vibrio-like field strains of German origin were isolated culturally from diseased domesticated ducks, muscovy ducks and geese, and were compared with reference strains NCTC 8443 (type strain) and NCTC 11170 of Vibrio metschnicovii using classical phenotypic and chemotaxonomic tests. Some V. cholerae strains were included in the chemotaxonomic tests for comparative purposes. On the basis of the classical phenotypic characteristics studied and the numerical analysis of the whole-cell fatty acid patterns, the Vibrio-like field strains were identified as Vibrio metschnicovii. The identification tables and the database of the computer software of two commercial micro-identification kits (API-20 NE, ID-32 E) did not identify the field strains. Of the reference strains used, only NCTC 8443 was correctly identified by the ID-32 E software.

Phenotypic characteristics of Riemerella anatipestifer and similar micro-organisms from various hosts.

A total of 199 Riemerella anatipestifer (RA) and RA-like field strains isolated culturally from birds of 12 different species and from pigs were characterized using classical phenotypic and chemotaxonomic tests. The RA reference strain ATCC 11845 was included in the study. On the basis of the classical phenotypic characteristics studied and the numerical analysis of the whole-cell fatty acid patterns, the RA reference strain and 123 field isolates were assigned to the indole negative (IN) variant and 10 isolates to the indole positive (IP) variant of the species RA. The IN strains were isolated not only from poultry and free-living wild ducks, but also from pigs, guillemots and from a budgerigar and a herring gull. All the IP isolates were isolated from domestic ducks. One field strain from a chicken and one from a black-headed gull, which were distinguished from RA mainly by the negative a-glucosidase reaction and production of yellow pigment respectively, showed fatty acid methyl ester profiles chemotaxometrically different from those of RA. Another 64 field strains isolated from domesticated ducks, geese and muscovy ducks with signs and lesions very similar to those caused by RA were phenotypically and chemotaxometrically clearly different from RA and could not be classified to any of the known species. This possible bacterial pathogen is therefore given the preliminary designation of Riemerella-like (RA-L) taxon 1502.