Øystein Angen

DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA.

The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae. Optimal conditions were obtained with 300 ng DNA added per well and bound by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance of results were a maximum of 15% standard deviation, calculated as a percentage of the mean for four replicate micro-wells, and that DNA similarities were not significantly different in at least two independent experiments. The relationship between DNA similarities obtained by the micro-well method (y) and by the spectrophotometric method (x) was y = 0.534x+30.6, when these criteria had been applied to 23 pairs of strains of Actinobacillus species, avian [Pasteurella] haemolytica-like bacteria and Mannheimia species. The correlation (Pearson) between DNA similarities obtained by interchange of strains used for covalent binding and hybridization was 0.794. Significantly lower DNA similarities were observed by the spectrophotometric compared with the micro-well method for three pairs of hybridizations. After removal of these data, the relationship between DNA similarities obtained by the micro-well and spectrophotometric methods improved to y = 0.855x + 11.0. It was found that the accuracy and precision of the micro-well method was at the same level as that of the spectrophotometric method, but the labour and analysis time were reduced significantly. The use of hybridization in the micro-well format will allow DNA-DNA hybridizations to be carried out between all strains selected for a particular taxonomic study, in order to construct complete data matrices and improve species definition.

Respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response.

Abstract Trans-tracheal aspirations from 56 apparently healthy calves and 34 calves with clinical signs of pneumonia were collected in six different herds during September and November 2002. The 90 samples were cultivated and investigated by PCR tests targeting the species Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Mycoplasma dispar, and Mycoplasma bovirhinis. A PCR test amplifying the lktC-artJ intergenic region was evaluated and shown to be specific for the two species M. haemolytica and Mannheimia glucosida. All 90 aspirations were also analyzed for bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus, and bovine corona virus by antigen ELISA. Surprisingly, 63% of the apparently healthy calves harbored potentially pathogenic bacteria in the lower respiratory tract, 60% of these samples contained either pure cultures or many pathogenic bacteria in mixed culture. Among diseased calves, all samples showed growth of pathogenic bacteria in the lower respiratory tract. All of these were classified as pure culture or many pathogenic bacteria in mixed culture. A higher percentage of the samples were positive for all bacterial species in the group of diseased animals compared to the clinically healthy animals, however this difference was only significant for M. dispar and M. bovirhinis. M. bovis was not detected in any of the samples. BRSV was detected in diseased calves in two herds but not in the clinically healthy animals. Among the diseased calves in these two herds a significant increase in haptoglobin and serum amyloid A levels was observed compared to the healthy calves. The results indicate that haptoglobin might be the best choice for detecting disease under field conditions. For H. somni and M. haemolytica, a higher percentage of the samples were found positive by PCR than by cultivation, whereas the opposite result was found for P. multocida. Detection of P. multocida by PCR or cultivation was found to be significantly associated with the disease status of the calves. For H. somni a similar association with disease status was only observed for cultivation and not for PCR.

Genomic relationships between selected phage types of Salmonella enterica subsp. enterica serotype typhimurium defined by ribotyping IS200 typing and PFGE

The genomic relationship between isolates representing 17 definitive phage types (DTs) of Salmonella enterica subsp. enterica serotype typhimurium (S. typhimurium) were analysed using three different typing methods: IS200 typing using the restriction enzymes EcoRI and PvuII, ribotyping using SmaI and EcoRI, and PFGE using XbaI. These methods were used to study four DTs in greater detail; in all 18 (DT 49), 10 (DT 110), five (DT 120) and seven (DT 135) isolates were studied. The combined data generated two large clusters, which could be divided into five groups. Within the first cluster, a close similarity was indicated between isolates of the following phage types: group A-DTs 44, 49, 135 and 204c, with DT 9 distantly related; group B-DTs 95 and 99; and group C-DTs 104a, 110 and 120. The other large cluster contained group D-DTs 10, 20 and 146, with DT 12 distantly related, and group E-DTs 69, 103 and 153. The same grouping was observed by principal component analysis, but a minimum spanning tree linked DT 12 to group E and not group D in this analysis. Among the typing methods used, IS200 gave the best representation of the overall similarity between the S. typhimurium isolates. Five different IS200 profiles were obtained among isolates belonging to DT 49. Only one profile was observed within each of the phage types DT 110, 120 and 135. All isolates within each of these four phage types were of one ribotype. Isolates of DT 49 showed four PFGE patterns, while one pattern was present within isolates of the three other phage types. Members of these four phage types were found to be clonally related as they formed tight subclusters separated from isolates of other phage types.

Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures.

Based on the 16S rRNA sequences of a collection of well-characterized strains of Haemophilus somnus a set of primers was selected as candidates for a species-specific PCR test. All investigated H. somnus strains were found positive in the test, including 12 strains earlier found to represent H. somnus by DNA-DNA hybridization as well as representatives of the 16 ribotypes previously described within this species. The specificity of the test was evaluated on a broad collection of strains within the family Pasteurellaceae and on other Gram positive and negative species. None of these strains gave rise to an amplicon in the PCR test. The performance of the test on mixed cultures was evaluated by adding P. multocida to serial dilutions of H. somnus and incubating the agarplates for 1 and 2 days. This showed that the PCR test applied to the harvest from an agarplate can be expected to detect a single colony of H. somnus in the presence of 10(9) CFU of P. multocida even after 2 days of incubation. In conclusion, the present PCR test has been shown to represent a specific test for identification of H. somnus both in pure and mixed cultures. It represents a quick, sensitive and reliable method for identification of bacteria belonging to this phenotypically heterogeneous and often slow growing species.

Final classification of Bisgaard taxon 9 as Actinobacillus arthritidis sp. nov. and recognition of a novel genomospecies for equine strains of Actinobacillus lignieresii

Phenotypic characterization of bacteria from diseased and healthy horses identified 18 isolates as Bisgaard taxon 9 and 11 isolates as Actinobacillus lignieresii. All strains of taxon 9 were alpha-galactosidase- and raffinose-positive and showed variable fermentation of (+)L-arabinose and (-)D-sorbitol. Strains of A. lignieresii were negative for these characteristics, with the exception of raffinose. Two strains from the (-)D-sorbitol-negative group of taxon 9 showed a 16S rRNA similarity of 99-6%, while 99.5% similarity was found between two strains of the (-)D-sorbitol-positive group. DNA-DNA hybridization between the two strains representing the (-)D-sorbitol-negative group showed 98% binding, and their closest relationship was to a strain of A. lignieresii (64%). The two strains of the (-)D-sorbitol-positive group showed 83% binding and were related to the (-)D-sorbitol-negative group at a 76% DNA binding level. Actinobacillus arthritidis sp. nov. is proposed for 12 strains of the (-)D-sorbitol-positive group. Actinobacillus genomospecies 2 is suggested for the six strains of the (-)D-sorbitol-negative group. Phenotypically, strains of A. arthritidis and Actinobacillus genomospecies 2 differ in (-)D-sorbitol fermentation and can be separated from Actinobacillus equuli by being trehalose-negative, while a positive reaction for alpha-galactosidase separates the taxa from A. lignieresii. The type strain of A. arthritidis, CCUG 24862T, was isolated from a joint of a horse. Three equine isolates of A. lignieresii that could not be separated from the type strain by means of phenotypic characteristics showed 98.6-100% 16S rRNA similarity, but only 96.4-96.7% similarity to the type strain. DNA-DNA hybridization between two strains of this group showed 92% binding but only 70% binding to the type strain of A. lignieresii. Consequently, these equine isolates of A. lignieresii represent a new genomospecies of Actinobacillus, suggested as genomospecies 1 because phenotypic characteristics are not presently available to separate it from the type strain of A. lignieresii.

Evaluation of a Multiplex PCR Test for Simultaneous Identification and Serotyping of Actinobacillus pleuropneumoniae Serotypes 2, 5, and 6

ABSTRACT Serotype-specific DNA regions involved in the biosynthesis of capsular polysaccharides (cps region) were used to develop a multiplex PCR test for the simultaneous species identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6. Primers specific for serotypes 2, 5, and 6 were combined with the already existing species-specific primers used in a PCR test based on the omlA gene. The PCR test was evaluated with serotype reference strains of A. pleuropneumoniae as well as 182 Danish field isolates previously serotyped by latex agglutination or immunodiffusion. For all serologically typeable strains, a complete correspondence was found between the results obtained by the multiplex PCR test and the results obtained by the traditional serotyping methods. Six of eight serologically nontypeable strains could be allocated to a serotype on the basis of the multiplex PCR results. The species specificity of the assay was evaluated with a collection of 93 strains representing 29 different species within the family Pasteurellaceae, as well as species normally found in the respiratory tracts of swine. All of these strains were negative by the multiplex PCR test, including 50 field isolates of the phylogenetically closely related species Actinobacillus lignieresii. When the multiplex PCR test was used to test Danish field strains, it was able to identify the serotypes of approximately 94% of all strains isolated from swine with clinical disease. More than 90% of the isolates that cross-reacted by the latex agglutination test were of serotype 2, 5, or 6. Determination of the serotype by PCR represents a convenient and specific method for the serotyping of A. pleuropneumoniae in diagnostic laboratories.

A retrospective study on salmonella infection in Danish broiler flocks

Abstract A retrospective longitudinal study was conducted to identify risk factors associated with Salmonella enterica infection in Danish broiler production. The study was based on information in the antemortem database (AM database) where data were available for all broiler flocks slaughtered over the 2-year period from 1992 to 1993 in Denmark. The AM database contains information collected by the ante-mortem veterinarians, from the slaughterhouses, and from the salmonella examinations carried out at the National Veterinary Laboratory. The epidemiological unit was the individual broiler flock. The salmonella status of the flock was determined by examining the caecal tonsils from 16 3-week-old chickens from each flock. This procedure would detect a salmonella-infected flock, with a probability above 95%, if the prevalence is above 20%. Furthermore, the structure and quality of the collected data have been evaluated. Fourteen variables were selected for analysis by multivariable logistic regression. An increased risk of salmonella infection in the broiler flocks was associated with the biggest hatcheries and feedmill, with an increasing number of houses on the farm, if the preceding flock was infected, and if the flock was reared in the autumn. Additionally, the main variables of the model were analysed by including a random effect at the house level. This resulted only in minor changes of the parameter estimates.

Investigations on the species specificity of Mannheimia (Pasteurella) haemolytica serotyping.

Eleven serotypes (1, 2, 5-9, 12-14 and 16) have been demonstrated within Mannheimia haemolytica. Subsequent serotyping of 166 Mannheimia haemolytica-like strains, genetically and phenotyphically distinct from Mannheimia haemolytica, and isolated from ruminants, pigs, hares and rabbits showed that 13.2% were typeable, 19 of which were serotype 11 representing strains now being classified as M. glucosida. In addition, three strains belonged to serotypes 6, 9 and 16, respectively. Additionally, the serotyping results of 98 (P.) haemolytica-like isolates from non-ruminant sources collected by the UK Veterinary Investigation Centres during the period 1982-1996 were investigated. None of these isolates have been kept, making further genetic characterization impossible. Among these isolates, 25.5% were typeable representing serotypes 1, 2, 3, 5, 6, 9, 10, 13 and 15. Substantial evidence has been reported indicating that M. haemolytica-like isolates from non-ruminant sources represent species different from M. haemolytica. The present investigation underlines that serotyping does not represent a reliable method for the identification of M. haemolytica or M. glucosida. These observations emphasize that extended phenotypic and genetic characterization is necessary for the proper identification of these organisms.

Relationships among strains classified with the ruminant Pasteurella haemolytica-complex using quantitative evaluation of phenotypic data.

The phenotypic relationships among 246 trehalose-negative strains classified under the [Pasteurella] haemolytica-complex in ruminants were investigated by clustering and multidimensional ordinations based upon 79 phenotypic characters. A quantitative evaluation of phenotypic data using a 5-level scoring system is presented permitting a comprehensive utilization of the recorded phenotypic variation among the strains in the analyses. Clustering and ordination analyses display complementary aspects of data which has been clearly demonstrated in this investigation. The main clusters revealed by the numerical techniques could be related to distinctive phenotypic differences and showed an extensive correlation with the recognized biogroups. This classification was based only upon 4 characters (fermentation of L-arabinose, D-sorbitol, glucosides and ornithine decarboxylase). In contrast, there was no obvious interpretation of the clusters formed by using binary scores. Phenotypic subgroups within the recognized biogroups have been described as well as a new, related group of bacteria, tentatively named Bisgaard taxon 36. Quantitative interpretation of phenotypic data seems to represent a promising method for finding relations among affiliated strains of bacteria and to assist in forming hypotheses for subsequent genotypic investigations.

Transmission of Actinobacillus pleuropneumoniae in pigs under field-like conditions: emphasis on tonsillar colonisation and passively acquired colostral antibodies.

The objectives of this study were to elucidate at which age tonsillar colonisation by Actinobacillus pleuropneumoniae occurs in pigs and relate this occurrence to the presence of colostral antibodies to A. pleuropneumoniae. The infection patterns were studied in an isolated cohort of pigs, which consisted of the offspring from five sows originating from a conventional pig herd. The sows were transferred to isolated research facilities before farrowing. A. pleuropneumoniae was detected on the tonsils of all sows. After a nursing period of 3 weeks, the pigs were weaned and reared isolated from other pigs until slaughter. The pigs were examined repeatedly for the presence of A. pleuropneumoniae on the tonsils and for antibodies to A. pleuropneumoniae using bacteriological and serological techniques, respectively.A. pleuropneumoniae was detected in the tonsils of one pig as early as 11 days after birth, showing that A. pleuropneumoniae can be transmitted from sow to offspring during a 3-week nursing period. The cumulative proportion of pigs carrying A. pleuropneumoniae in their tonsils increased significantly between the age of 4-12 weeks. This age period corresponded to the age at which the proportion of pigs with detectable levels of colostral antibodies to the different serotypes of A. pleuropneumoniae was declining. Since these two events take place in the same age period, we expect a possible biological association between the level of the passive immunity and the degree of tonsillar colonisation. The median duration of tonsillar colonisation was estimated to approximately 7-8 weeks.