Magne Bisgaard

Prevalence of gastrointestinal helminths in different poultry production systems

A cross-sectional prevalence study of gastrointestinal helminths in Danish poultry production systems was conducted on 268 adult chickens selected at random from 16 farms in Denmark from October 1994 to October 1995. The trachea and the gastrointestinal tract of each bird was examined for the presence of helminths. In the free-range/organic systems the following helminths were found: Ascaridia galli (63.8%), Heterakis gallinarum (72.5%), Capillaria obsignata (53.6%), Capillaria anatis (31.9%) and Capillaria caudinflata (1.5%). In the deep-litter systems: A. galli (41.9%), H. gallinarum (19.4%) and C. obsignata (51.6%). In the battery cages: A. galli (5%) and Raillietina cesticillus or Choanotaenia infundibulum (3.3%). Exact identification of the cestodes was not possible because of missing scolexices. In the broiler/parent system: C. obsignata (1.6%), and finally for the backyard system: A. galli (37.5%) H. gallinarum (68.8%), C. obsignata (50.0%), C. anatis (56.3%) and C. caudinflata (6.3%). The results confirm the higher risk of helminth infections in free-range and backyard systems but prevalence may also be high in deep litter systems.

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.

Ecology and significance of Pasteurellaceae in animals.

The reservoir of eighty-one taxa/groups classified with the family Pasteurellaceae Pohl 1981 is reviewed based upon published data and own investigations. With the exception of certain strains of P. multocida, A. pleuropneumoniae and [H.] paragallinarum organisms belonging to this family are usually regarded as opportunistic, secondary invaders which under normal conditions coexist peacefully with the animal host on mucosal membranes of the upper respiratory- and lower genital tracts. Very little is known about factors that govern the ecological preferences that certain members of this family show for specific surfaces and hosts. Mechanisms of colonization, survival and multiplication, invasion and pathogenic action are incompletely understood. The significance of Pasteurellaceae in animals and man has recently been reviewed. Subsequent publications have underlined the significance of biovars 2 of P. canis and P. avium and ornithine negative P. multocida in pneumonia in cattle. In addition, differences in pathogenicity have been demonstrated for different serovars of [H.] parasuis. The disease potential of many taxa/groups is only incompletely known.

Ribotypes of Salmonella enterica serovar Gallinarum biovars gallinarum and pullorum

Ninety-four isolates of Salmonella enterica serovar Gallinarum biovar pullorum and forty-one isolates of biovar gallinarum were ribotyped using the enzymes, HindIII, EcoRI and SmaI, and a digoxigenin-labelled E. coli-derived rRNA probe. Using HindIII, 13 profile types were observed within biovar gallinarum and 12 within biovar pullorum. The most common types accounted for 39% of biovar pullorum isolates and 47% for biovar gallinarum. EcoRI digests revealed two profile types within biovar pullorum, one accounting for 96% of the isolates, and three EcoRI profiles within biovar gallinarum, with 81% of the isolates belonging to the dominant type. Using SmaI, biovar pullorum showed two profile types with 94% of the isolates belonging to the dominant one, while SmaI digestion revealed only one ribotype within biovar gallinarum. The SmaI ribotype of biovar gallinarum was identical to the less common of the two SmaI types in biovar pullorum. Two of the HindIII profile types were seen in both biovars. The two biovars did not share any of the EcoRI profiles. Numerical analysis based on the ribotypes revealed a 94% similarity between the two biovars, but they clustered separately in a similarity dendrogram, underlining the existence of two different biovars. The results indicate that ribotyping, especially using EcoRI, may be useful in separating biovars gallinarum and pullorum. The ribotypes obtained with isolates from different countries indicated that different clones of biovar gallinarum might exist in different regions.

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.

Tissue distribution of haemolytic Gallibacterium anatis isolates in laying birds with reproductive disorders.

Gallibacterium anatis biovar haemolytica has been suggested to have a causal role in peritonitis and salpingitis in chickens. Therefore, the aim of this study was to investigate the occurrence of G. anatis biovar haemolytica in chickens with reproductive disorders. One hundred and forty one birds from 31 layer flocks were submitted for necropsy and the following organs were examined for bacteria: choana, trachea, lung, heart, liver, spleen, ovary, oviduct, duodenum and cloaca. Examination for Escherichia coli was included as it can induce reproductive disorders. G. anatis was isolated in pure culture from the reproductive tract of affected birds in six of the 31 flocks while E. coli was obtained in pure culture from 10 of them. Both G. anatis and E. coli were isolated from the reproductive tract of 14 of the 31 flocks. The genetic diversity of the Gallibacterium isolates was assessed by amplified fragment length polymorphism on a subset of 83 isolates. Generally, each flock was infected with a single clone, which could be isolated from various sites in the birds. However, in two flocks, the majority of birds yielded positive samples from the internal organs, indicating that these particular clones may be more invasive. The findings support previous suggestions that G. anatis biovar haemolytica is associated with infection of the reproductive tract of chickens, making it a likely cause of lowered productivity and an animal welfare concern.

High Diversity of Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Italian Broiler Flocks

ABSTRACT We characterized 67 Escherichia coli isolates with reduced susceptibility to cefotaxime or ceftiofur obtained from healthy broilers housed in five Italian farms. The blaCTX-M-1, blaCTX-M-32 and blaSHV-12 β-lactamase genes were identified on IncI1, IncN, or IncFIB plasmids. Considerable genetic diversity was detected among the extended-spectrum β-lactamase (ESBL)-producing isolates, and we identified indistinguishable strains in unrelated farms and indistinguishable plasmids in genetically unrelated strains. The detection of highly mobile plasmids suggests a potential animal reservoir for β-lactamase genes.

The effect of concurrent infections with Pasteurella multocida and Ascaridia galli on free range chickens

Pasteurella multocida and Ascaridia galli are observed with high prevalences in free range chickens in Denmark, but the impact is unknown. A study was carried out to examine the interaction between A. galli and P. multocida in chickens and the impact on production. Five groups, each with 20 18-week-old Lohmann Brown chickens were infected. Group 1 was orally infected with 1000+/-50 embryonated A. galli eggs. Group 2 received 10(4) cfu P. multocida intratracheally. Group 3 was infected with A. galli and subsequently with P. multocida. Group 4 was infected with P. multocida followed by A. galli. Group 5 was the control. The study ran for 11 weeks where clinical manifestations, weight gain and egg production were recorded. Excretion of P. multocida was determined on individual basis and blood smears were made for differential counts. At the end of the study pathological lesions and the number of adult worms, larvae and eggs in the faeces were recorded. The birds were more severely affected when infected with both pathogens compared to single infections with A. galli or P. multocida, respectively. A lower weight gain and egg production was observed with dual infections. A. galli infection followed by a secondary P. multocida infection resulted in more birds with pathological lesions and continued P. multocida excretion. In conclusion a negative interaction between A. galli and P. multocida was observed and it is postulated that free range chickens are at higher risk of being subjected to outbreaks of fowl cholera when they are infected with A. galli.

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.

Porcine-origin gentamicin-resistant Enterococcus faecalis in humans, Denmark.

During 2001–2002, high-level gentamicin-resistant (HLGR) Enterococcus faecalis isolates were detected in 2 patients in Denmark who had infective endocarditis and in pigs and pork. Our results demonstrate that these isolates belong to the same clonal group, which suggests that pigs are a source of HLGR E. faecalis infection in humans.