Studies on the prevalence of Riemerella anatipestifer in the upper respiratory tract of clinically healthy ducklings and characterization of untypable strains.

A total of 44 bacterial strains obtained from 49 clinically healthy ducklings of different ages originating from four different farms were identified as members of the species Riemerella anatipestifer (RA) using conventional biochemical test methods. Numerical analysis of the whole-cell fatty acid patterns of these isolates resulted in two different clusters, one of which showed a similar pattern to that of the type strain of RA. Strains having a different fatty acid methyl esters (FAME)-profile (cluster II) were designated R. anatipestifer-like (RA-like). Sequencing of 16S rRNA genes of RA-like field isolates revealed 99% identity to RA. The significance of these observations are discussed. The present findings document for the first time that RA seems to represent a normal part of the pharyngeal flora of healthy Pekin ducks.

Competitive exclusion of Salmonella enteritidis by Salmonella gallinarum in poultry.

Salmonella Enteritidis emerged as a major egg-associated pathogen in the late 20th century. Epidemiologic data from England, Wales, and the United States indicate that S. Enteritidis filled the ecologic niche vacated by eradication of S. Gallinarum from poultry, leading to an epidemic increase in human infections. We tested this hypothesis by retrospective analysis of epidemiologic surveys in Germany and demonstrated that the number of human S. Enteritidis cases is inversely related to the prevalence of S. Gallinarum in poultry. Mathematical models combining epidemiology with population biology suggest that S. Gallinarum competitively excluded S. Enteritidis from poultry flocks early in the 20th century.

Quinupristin/dalfopristin-resistant enterococci of the satA (vatD) and satG (vatE) genotypes from different ecological origins in Germany.

The semisynthetic streptogramin combination quinupristin/dalfopristin (Synercid) is a promising alternative for treatment of infections due to multiply resistant gram-positive bacteria including vancomycin-resistant Enterococcus faecium. Resistance is mediated by acetyltransferases SatA (VatD) or SatG (VatE). Recent papers have indicated a possible link between the use of the streptogramin virginiamycin S/M as a feed additive in commercial animal husbandry and a selection of quinupristin/dalfopristin-resistant E. faecium (QDRE). We screened manure samples from two different turkey farms and from six different pig farms (using virginiamycin), samples from a sewage water treatment plant, 24 broiler carcasses, 10 pork samples, and 200 stool samples of nonhospitalized humans for QDRE. Our strain culture collection of hospital E. faecium isolates from the last 2 years was also reviewed for QDRE. All manure and sewage samples were positive for QDRE, as well as 11 from broiler carcasses (46%), 1 from pork (10%), and 28 from human stool specimens (14%). Thirty-six hospital isolates of E. faecium exhibited resistance to quinupristin/dalfopristin. In 141 QDRE of different origin satA (vatD) and satG (vatE) genes were detected (seven isolates from humans with an unknown resistance mechanism). Streptogramin resistance determinants were tansferable in filtermating experiments for 5 of 10 satA (vatD) and 9 of 22 satG (vatE) isolates. Different EcoRI patterns of satG (vatE) plasmids and corresponding hybridizations of the satG (vatE) gene indicated nonhomologous resistance plasmids in isolates of different origin. The results of this study indicate a common gene pool for streptogramin resistance in E. faecium of different ecological origin. A selection of QDRE using the streptogramin virginiamycin S/M as a feed additive and a spread of the resistance via the food chain to humans is probable.

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.

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.

Biotin-streptavidin enzyme-linked immunosorbent assay for the detection of antibodies to Campylobacter jejuni and C. coli in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.

Coenonia anatina gen. nov., sp. nov., a novel bacterium associated with respiratory disease in ducks and geese.

Taxon 1502 was originally described as a Riemerella anatipestifer-like bacterium causing exudative septicaemia in ducks and geese. In the present study, an integrated genotypic and phenotypic approach was used to elucidate the phylogenetic affiliation and taxonomic relationships of 12 strains of taxon 1502. Whole-cell protein and fatty acid analyses and an extensive biochemical examination by using conventional tests and several API microtest systems indicated that all isolates formed a homogeneous taxon, which was confirmed by DNA-DNA hybridizations. 16S rDNA sequence analysis of a representative strain (LMG 14382T) indicated that this taxon belongs to the Cytophaga-Flavobacterium-Bacteroides phylum and revealed a moderate but distinct relationship to species of the genus Capnocytophaga (overall 16S rDNA sequence identities were 88.8-90.2%). Taxon 1502 is concluded to represent a single species that should be allocated to a novel genus, and the name Coenonia anatina gen. nov., sp. nov. is proposed. The DNA G + C content of representative strains was 35-36 mol% and the type strain is LMG 14382T.

Experiences with multispecies polymerase chain reaction and specific oligonucleotide probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

Amplified fragments of the rDNA coding for 16S rRNA of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) were blotted on nylon membranes, followed by dot-blot detection with two species-specific digoxigenin-(DIG)-labeled oligonucleotide probes. The sensitivity and specifity of the tests were determined in titration studies with purified homologous and heterologous DNA. With the detection protocol used, the MSYV8/31 probe showed 100% specifity for MS, while both MG and the related species Mycoplasma imitans were recognized by the MGAV8/31 probe. Both DIG-labeled oligonucleotides gave positive results in the colorimetric assay with 10 to 100 ng homologous non-amplified DNA and polymerase chain reaction (PCR) amplificates of 100 fg homologous template DNA. There was no reaction with heterologous strains when amplificates starting with a 106-fold amount of template DNA (100 ng) were tested in dot-blots. The suitability for field samples was demonstrated with tracheal swabs from turkeys and chickens, and the results were compared with mycoplasma growth in cultures of the same swabs. Both tests had an accuracy of over 95%, a high sensitivity and specificity, and high predictive values of positive or negative results. There was no significant difference between the results obtained by the two methods. PCR in combination with dot-blotting is a relatively simple method for the detection of mycoplasma infections, and a valuable extension of current diagnostic tools.

Identification of members of the Pasteurellaceae isolated from birds and characterization of two new taxa isolated from psittacine birds.

A collection of 43 unclassified members of the Pasteurellaceae, most of which were obtained from lesions, were investigated using an extensive battery of phenotypical tests as well as by ribotyping. The isolates had been made from Anser anser forma (f) domestica (d), Agapornis fischer, Amazona spp., Ara macao, Columba livia f.d., Melanopsittacus undulatus, and Psittacus erithacus. Comparison of results with those obtained from reference strains allowed classification of 25 strains. Three strains were identified as Pasteurella dagmatis, P. sp. A, and [P.] aerogenes, respectively. Twenty strains were classified as taxon 3 and two as taxon 14. Eighteen strains, all originating from psittacine species, belonged to two new taxa, tentatively named Bisgaard taxon 33 and taxon 37. Characters obtained with taxon 33 allowed classification within the family Pasteurellaceae, while the final classification of taxon 37 remains to be investigated. The present investigation underlines the problems confronting diagnostic laboratories attempting to identify members of the family Pasteurellaceae isolated from birds.

Pelistega europaea gen. nov., sp. nov., a bacterium associated with respiratory disease in pigeons: taxonomic structure and phylogenetic allocation.

Twenty-four strains isolated mainly from infected respiratory tracts of pigeons were characterized by an integrated genotypic and phenotypic approach. An extensive biochemical examination using conventional tests and several API microtest systems indicated that all isolates formed a phenotypically homogeneous taxon with a DNA G + C content between 42 and 43 mol%. Whole-cell protein and fatty acid analysis revealed an unexpected heterogeneity which was confirmed by DNA-DNA hybridizations. Four main genotypic sub-groups (genomovars) were delineated. 16S rDNA sequence analysis of a representative strain indicated that this taxon belongs to the beta-subclass of the Proteobacteria with Taylorella equigenitalis as its closest neighbour (about 94.8% similarity). A comparison of phenotypic and genotypic characteristics of both taxa suggested that the pigeon isolates represented a novel genus for which the name Pelistega is proposed. In the absence of differential phenotypic characteristics between the genomovars, it was preferred to include all of the isolates into a single species, Pelistega europaea, and strain LMG 10982 was selected as the type strain. The latter strain belongs to fatty acid cluster I and protein electrophoretic sub-group 1, which comprise 13 and 5 isolates, respectively. It is not unlikely that the name P. europaea will be restricted in the future to organisms belonging to fatty acid cluster I, or even to protein electrophoretic sub-group 1, upon discovery of differential diagnostic features.

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.

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.

A comparison of a commercial PCR-based test to culture methods for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in concurrently infected chickens.

The suitability of commercial PCR-based test kits for the detection of either Mycoplasma gallisepticum (MG) or M. synoviae (MS) was compared to detection by culture. The MG and MS kit detected six and five homologous strains respectively in broth cultures and there were no reactions with thirteen het-erologous species including M. imitans, a species phylogenetically closely related to MG. Tracheal and lung/air-sac swabs were collected from twenty 17-week-old commercial pullets which were seropositive for MS and were compared for detection of MS by kit and by culture. The results were in agreement for 13 positive and 22 negative swabs, while the remaining five swabs were either PCR-positive only (two) or culture-positive only (three). Tracheal swabs taken from seventy-six 31-week-old layers from a MS seropositive commercial flock which had been experimentally infected with MG were used to compare the MG DNA probe kit and culture. Of 76 swabs 39 were MG positive and 12 were negative by both methods. MG was detected by PCR test only in 23 other specimens, while only two swabs were negative by PCR but positive by culture. The difference between the detection methods was significant (McNemar test, P < 0.001). Concurrent MS infection was detected by the PCR-based kit in 45 of these hens.

[Pilot study on the prevalence of Ornithobacterium rhinotracheale infections in food chickens in northwest Germany]. / Pilotstudie zur Prävalenz der Ornithobacterium rhinotracheale-Infektion bei Masthühnern in Nordwestdeutschland.

Samples of sera from broiler chicken and broiler chicken breeder flocks of North Western Germany were examined for presence of antibodies against Ornithobacterium (O.) rhinotracheale with a self-developed ELISA system. A total of 3600 samples of sera from 190 broiler flocks in 70 different farms were tested. In 332 samples of sera (9.4%) from 25 flocks (13.2%) in 22 farms specific antibodies were detected. Additionally, serum samples from the breeder hens, taken between the 9, and 61, weeks of age were tested. In 28 from 29 flocks (96.6%) specific antibodies were detected using this ELISA-system. First detectable antibodies were found between the 14, and 36, week of age in the parent breeder flocks. These results show that infections with O. rhinotracheale are widely distributed in the parent-breeder chicken broiler flocks. The significance of this bacterium as the cause of respiratory diseases in chickens are discussed.

Bordetella trematum sp. nov., isolated from wounds and ear infections in humans, and reassessment of Alcaligenes denitrificans Rüger and Tan 1983.

Ten strains recognized on the basis of a computer-assisted numerical comparison of whole-cell protein patterns as members of a novel species belonging to the family Alcaligenaceae were examined by using an integrated phenotypic and genotypic approach. This species, for which we propose the name Bordetella trematum sp. nov., was more closely related to the type species of the genus Bordetella (Bordetella pertussis) than to the type species of the genus Alcaligenes (Alcaligenes faecalis) and had the general characteristics of members of this family (i.e., a DNA base ratio in the range from 57 to 70 mol%, a fatty acid profile characterized by high percentages of 16:0, 17:0 cyclo, and 14:0 3OH, nonsaccharolytic metabolism, and several classical biochemical characteristics, including aerobic and microaerobic growth, catalase activity, assimilation of citrate, an absence of anaerobic growth, and an absence of acetylmethylcarbinol and indole production, gelatin liquefaction, and esculin hydrolysis). A reevaluation of the criteria used to classify Alcaligenes denitrificans Rüger and Tan 1983 and Achromobacter xylosoxidans Yabuuchi and Ohyama 1971 as subspecies of Alcaligenes xylosoxidans and additional evidence provided in recent studies revealed that, consistent with present standards, it is appropriate to consider these two taxa distinct species of the genus Alcaligenes.

Differentiation of Salmonella gallinarum and Salmonella pullorum by their whole-cell fatty acid methyl ester profiles.

The whole-cell fatty acid methyl ester (FAME) profiles of Salmonella (S.) gallinarum and Salmonella (S.) pullorum strains were compared in the computer-linked gas-liquid chromatography. The profiles of whole cellular FAMEs allow the separation and identification S. gallinarum and S. pullorum by the Microbial Identification System and so can be used for their differentiation.

[Prevalence of motile salmonellae in egg-laying hens at the end of the laying period]. / Prävalenz beweglicher Salmonellen in Legehühnern am Ende der Legeperiode.

A total of 3504 hens of the layer-type from 122 flocks (belonging to 89 farms), each with more than 10,000 animals, were culturally examined at the time of slaughter. Of these hens, 2112 (60.3%) from 74 flocks (60.7%) were obtained from 21.3% of the laying-hen farms in a selected region of Lower Saxony in Germany. The other hens came from the remaining part of Lower Saxony and seven other German states (Brandenburg, Mecklenburg Vorpommern, North Rhine Westphalia, Schleswig Holstein, Saxony, Saxony Anhalt, and Thuringia). After arrival at the slaughter house, a random sample of 29 layers was collected from each of the flocks, and liver and spleen, as well as cecal samples, were separately cultured for each bird. Motile salmonellae could be proved in 365 (10.4%) layers from 67 flocks (54.9%). In the selected region, 48 out of 74 flocks (64.9%) and 289 out of 2112 layers (13.7%) were Salmonella-positive. However, the isolation frequency of salmonellae did not differ significantly between flocks of brown and white layers. These Salmonella (S.) isolates could be serologically assigned to 6 different serovars, namely S. enteritidis (SE), S. infantis (SI), S. livingstone (SL), S. typhimurium (ST), S. indiana (SID) and S. cerro; only one isolate of serogroup D1 was incompletely serotyped. SE was detected in 5.8% of the hens from 47.5% of the tested flocks, of which 4.6% of the animals and 32.8% of the flocks came from the selected region in Lower Saxony. The SE isolates were classified into 12 different lysotypes. In 41 out of 58 SE-positive flocks (70.7%), the isolates belonged to lysotype (lt) 4, in 12 flocks (20.7%) to lt 8, in 5 flocks (8.6%) to lt 7, and in 3 flocks (5.2%) to lt 11. A total of 190 (93.1%) out of 204 isolates of the serovar SE carried plasmids. All the plasmid-positive SE-strains harboured the serovar-specific 37 MD virulence-plasmid, nine of them (4.4%) in conjunction with a second and eight strains (3.9%) with a second and a third smaller plasmid.

[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.