Search

The serotypes of Pasteurella multocida were predicted based on whole genomic sequences (WGS) with specific genes of the capsular and liposaccharide (LPS) outer core polysaccharide regions as targets. A total of 56 strains were whole genomic sequenced and in addition all assembled genomes from NCBI were included for comparison. BIGSdb (Bacterial Isolate Genome Sequence Database) was installed on a Linux server and targets for capsular types A, B, D, E and F were defined as gene sequences of hyaD, bcbD, dcbF, ecbJ and fcbD, respectively and targets for LPS groups 1, 2, 3, 4, 5, 6, 7 and 8 were defined as gene sequences of pcgB, nctA, gatF, latB, rmlA, nctB, ppgB and natG, respectively. The serotypes of P. multocida were predicted from WGS by designating the capsular type and LPS group as well as subtype alleles to isolates. Comparisons between WGS predictions of capsular types and classical phenotypic typing showed correspondence in 87 % of cases whereas comparisons of WGS predictions of LPS groups to phenotypic typing corresponded for 82 % of the strains. In total 93 % and 94 % of the strains available with WGS could be capsular and LPS group typed, respectively. The server is free to access from https://ivsmlst.sund.ku.dk., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of the investigation was to predict the serotypes of M. haemolytica based on whole genomic sequences with the capsular gene region as target. A total of 22 strains selected to have been serotyped and to represent all serotypes were investigated by whole genomic sequencing. The BIGSdb (Bacterial Isolate Genome Sequence Database) was downloaded and installed on a Linux server. Here the sequence database was setup with unique loci at serotype level. The server allows serotypes of M. haemolytica to be predicted from whole genomic sequences and the service is available to the public for free from https://ivsmlst.sund.root.ku.dk., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of the investigation was to develop a definitive typing system for Rodentibacter pneumotropicus. A total of 79 strains including the type strain of R. pneumotropicus, all associated with rodents were used to develop a multi-locus sequence typing scheme (MLST). Primers were designed for conserved regions of seven house-keeping genes (atpG, frdB, gdh, pgi, pmi, recA, zwf) and internal fragments of 399-839 bp were sequenced for all strains. The genes were also extracted in full length from whole genomic sequences of 14 strains of which 10 were sequenced in the current study. The number of alleles at the different loci ranged from 5 to 7 and a total of 20 allelic profiles or sequence types were recognized amongst the 79 strains. Analysis of the MLST data showed that some STs have been stable over many years probably circulating in the same colonies and probably transferred between colonies. We assume that this MLST scheme may provide a high level of resolution and might be an excellent tool for studying the population structure and epidemiology of R. pneumotropicus. Further development of the scheme is expected by including more genes and more strains and involve whole genomic sequencing., (Copyright © 2019. Published by Elsevier B.V.)

The aim of the study was to investigate cellulitis caused by Escherichia coli which has been responsible for economic and welfare problems in Danish broiler production between 2014 and 2016. The study included 13 flocks with unusually high condemnation rates due to cellulitis during a period of approximately one year. From six flocks, 126 condemned carcasses were collected at a Danish slaughterhouse. Further 272 broilers dead on their own were collected on nine broiler farms from flocks with increased mortality and cellulitis (2 farms included both birds from the rearing period and broilers subsequently condemned). All broilers were subjected to post mortem investigation including bacteriology and 247 E. coli isolates were obtained in pure culture from typical lesions of cellulitis. Two-hundred-thirty six E. coli isolates were investigated by pulsed field gel electrophoresis for clonality and 21 selected strains representing major clones were subsequently multi locus sequence typed allowing comparison to sequence types (ST) in the databases. One dominating PFGE type (A) was found to cause cellulitis on all 13 flocks (67% of all isolates). The clone belonged to ST117, which is well described as a pathogen in poultry, and was the primary agent responsible for cellulitis. Whole genome sequencing of eight E. coli isolates confirmed the close genetic relationship between isolates from the outbreaks and showed the presence of genes predicted to encode for the autotransporter proteins aatA, pic and upaG, reported to be of importance for adhesion of E. coli to eukaryotic cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In rabbit, P. multocida is considered a predominant pathogenic agent; despite this, few data on the molecular epidemiology are available so far. The aim of this work was to characterize P. multocida isolates from rabbit affected by various diseases in Italy. Comparison was made to reference strains from other countries. Thirty-nine isolates were tested using PCRs to detect the genes coding capsular antigens, virulence factors and lipopolysaccharide structures (LPS). Multilocus sequence typing (MLST) was performed and 19 STs registered that belonged to 9 clonal complexes. Italian isolates were all related to P. multocida subsp. P. multocida. Three sequence types dominated (ST9, ST50 and ST74). The isolates were assigned to capsular types A (20/39), D (9/39) and F (10/39), to virulence genes pfhA (13/39), hgbB (21/39) and pfhA+hgbB (4/39) (one without virulence factors) and the isolates either belonged to the LPS genotypes 3 (22/39) or 6 (17/39). The clonal relationships of the Italian strains from rabbit had similarity to previously reported rabbit isolates that belonged to ST9, ST74, ST204 and ST206, however, they differed from other rabbit references strains that belonged to six other STs. In particular, ST9 with capsular type F has been previously reported from diseased rabbit in Czech Republic and ST74 has been observed for older rabbit isolates. ST50 has probably been reported from Spain. ST9 and ST50 have previously also been reported from birds and pig, respectively, whereas ST74 has exclusively been reported from pig. It remains to be investigated if the isolates obtained from diseased rabbit in Italy represent introductions from other host or they are primarily of rabbit origin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, four broiler parent flocks have been followed from the onset of the production period (week 20) until slaughter (week 60). Every week, approximately ten dead broiler breeders, randomly selected among birds dead on their own, were collected and subjected to a full post mortem analysis including bacteriological examination. In total 997 breeders were investigated and for the first time Staphylococcus agnetis was isolated in pure culture from cases of endocarditis and septicemia from 16 broiler breeders. In addition, the cloacal flora from newly hatched chickens originating from the same four flocks were characterized and S. agnetis was found in pure culture of several newly hatched chickens (n=12) and only in one case in combination with another species. Clonality of the isolates was examined by pulsed-field-gel-electrophoresis which showed indistinguishable patterns in isolates from both broiler breeders and broilers. Three isolates were whole genome sequenced to obtain knowledge on virulence genes. The isolates harbored a number of genes encoding different fibrinogen binding proteins and toxins which might be important for virulence. The present findings demonstrate that S. agnetis may be associated with mortality in broiler breeders. No disease was associated with the broilers which were found positive for S. agnetis in the cloaca., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Escherichia coli is of major importance in industrial broiler production as the main cause of salpingitis and peritonitis in broiler breeders. Furthermore E. coli is the most common cause of first week mortality in broiler chickens. The aim of the present study was to investigate the transmission of E. coli, isolated from broiler breeders with salpingitis, to the progeny and the possibility of subsequent first week mortality. Four parent flocks were followed during the whole production period (20-60 weeks) by post mortem and bacteriological examination of randomly selected dead birds. Newly hatched chickens from each flock were swabbed in the cloaca on four occasions (parent age 30, 40, 50, 60 weeks) and E. coli was isolated. Causes of first week mortality were determined pathologically and bacteriologically. E. coli isolates from parents, newly hatched chickens and first week mortality were selected for Pulsed-Field-Gel-Electrophoresis (PFGE) and Multi-Locus-Sequence-Typing (MLST) to determine their clonal relationships. E. coli was the main cause of both salpingitis in parents and first week mortality in broilers, and E. coli dominated the bacterial flora of the cloaca of newly hatched chickens. PFGE of E. coli showed identical band patterns in isolates from the three different sources indicating a transmission of E. coli from parent birds to chickens. In conclusion, E. coli isolated from salpingitis in broiler parents were found to be transmitted to broilers in which some sequence types contributed to the first week mortality., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Two serovars of Pasteurella multocida, B:2 and E:2, have been reportedly associated with haemorrhagic septicaemia (HS), a peracute and devastating disease mainly affecting cattle and water buffaloes. We multilocus sequence typed (MLST) 64 isolates of P. multocida including 55 associated with HS and found that they mainly included sequence type (ST) 122 (n=50) and rarely ST63 (n=1), ST147 (n=2) and ST162 (n=2) compared to other members of the species isolated from other lesion types and hosts. Single-nucleotide polymorphisms suitable for specific detection of STs associated with HS were detected in the est gene. A new HS-est-RT-PCR (est indicating the target gene) specifically detected ST122, ST63, ST147 and ST162 associated with HS. The new HS-est-RT-PCR did not detect strains of ST151 with capsular type D isolated from pigs that were found positive with a previously published HS PCR detection method. The new HS-est-RT-PCR represents a fast and specific detection of the specific types of P. multocida involved in HS. The HS-est-RT-PCR developed in the current study seems to more accurately identify isolates of P. multocida associated with HS compared to PCR detection methods previously published., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A collection of 46 avian pathogenic Escherichia coli (APEC) isolates was examined for the presence of mutators by determining the rate of mutation to rifampicin resistance. The collection included 34 E. coli isolates obtained in pure culture from chronic lesions of salpingitis and peritonitis in 34 broiler breeders, of which 12 were associated with the development of secondary septicemia. Twelve additional isolates were obtained from a clonal outbreak (ST95) of E. coli peritonitis syndrome (EPS), the lesions of which changed gradually over time into a subacute/chronic form. The hypothesis of the present study was that mutation rates would be higher for chronic infection isolates than for isolates from acute infections/exacerbations. The distribution of mutation rates followed a pattern similar to that found for other clinical isolates of E. coli, with a modal/median value of 1.47 × 10(-8). Of the 46 isolates, 24% (n=11) were weakly hypermutable (2.00 × 10(-8) ≤ μ<2.00 × 10(-7)), however, no strong mutators were detected (μ ≥ 2.00 × 10(-7)). Chronic salpingitis isolates had the highest proportion (45%, P=0.001) of weak mutators and also, significantly higher mutation rates (P=0.003) compared to isolates that caused septicemia (4%). In addition, mutation rates were significantly lower among ST95 isolates (P<0.0005), and among isolates from the same clonal group as ST95 (P=0.027), when compared to isolates from other groups. Although a clear association with the time phase of infection (as lesions of EPS became more chronic) could not be observed (ρ=0.523, P=0.081), a higher frequency of weak mutators among chronic infection isolates suggests that increased mutation rates play a role in adaptation of APEC to long-term persistence in an infected host environment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Typhimurium phage type DT41 has previously been identified from salmonella-positive broiler breeder flocks in Denmark and isolates obtained from different flocks have demonstrated major diversity by multiple-locus variable-number tandem-repeats analysis (MLVA) typing. To elucidate whether the high diversity observed by MLVA was related to multiple independent introductions at farm level or genetic instability of markers, we investigated the genomic stability of different clones of S. Typhimurium DT41. In the in vitro genomic stability experiment, feed pellet- and dust samples inoculated with four strains of DT41 were kept at three different temperatures. The in vitro genomic stability was also assessed by conducting a serial passage experiment. In a subsequent in vivo experiment, broiler breeders of three different age groups were challenged with a strain of poultry and human origin, respectively. The in vitro experiment demonstrated that DT41 survived more than 6 months in feed-pellets at 20 °C whereas the survival in dust was less than 4 weeks. Infection pattern and excretion varied for the poultry and human strain and birds of different age groups as revealed by the in vivo experiment. Genetic stability of cultures obtained from the in vitro and in vivo survival/passage was investigated by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and MLVA. The results of plasmid profiling and PFGE demonstrated genomic stability of all but one strain kept in dust at 20 °C for 3 weeks. Minor genetic changes were observed in isolates from the in vitro experiment as revealed by MLVA. The epidemiological impact of these findings is briefly discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Neuraminidases (sialidases) are virulence factors of several poultry pathogens. Ornithobacterium rhinotracheale is a well known poultry pathogen causing respiratory disease in chickens and turkeys all over the world. We investigated whether O. rhinotracheale has neuraminidase enzymatic activity (NEAC). We tested NEAC in 47 O. rhinotracheale strains isolated from turkeys and chickens in eight countries. All strains showed relatively strong NEAC and considerable levels of NEAC were detected also in "cell-free supernatants" of their pelleted cells. Zymography using neuraminidase-specific chromogenic substrate indicated that a protein with molecular mass of ~40kDa and isoelectric point (pI) of ~8.0 is a putative neuraminidase of O. rhinotracheale. Notably, the genome of the type strain of O. rhinotracheale, DSM 15997 contains a gene (Ornrh&#95;1957) encoding a putative neuraminidase with such Mw (39.5 kDa) and pI (8.5). We sequenced a corresponding genomic region of 20 O. rhinotracheale strains and found five distinct types of the neuraminidase gene (termed nanO) sequences. Most diversified nanO sequence was found in two strains isolated from chickens in Hungary in 1995. Their nanO sequences differ from that of the type strain (LMG 9086(T)) in 27 nucleotides. O. rhinotracheale neuraminidase showed capacity to cleave sialic acid from chicken and turkey glycoproteins. It cleaved sialic acid from SAα(2-6)gal moiety of their serum proteins, including immunoglobulin G (IgG) and transferrin. O. rhinotracheale also desialylated chicken and turkey tracheal mucus glycoprotens with SAα(2-3)gal moieties. This study provides the first evidence that O. rhinotracheale has neuraminidase which can desialylate glycoproteins of its natural hosts., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The genetic relatedness and virulence profiles of avian pathogenic Escherichia coli that caused salpingitis and peritonitis in 68 broiler breeders from 21 Danish farms were determined by multilocus sequence typing (MLST), pulsed-field-gel-electrophoresis (PFGE), ECOR phylogrouping, and PCR-based virulotyping. Phylogroups A, B1, B2, and D accounted for 19.1%, 5.9%, 52.9%, and 22.1% of the isolates, respectively. Overall, a total of five main MLST-based clonal groups (3-38 isolates) were identified, comprising 85.3% of the isolates. The most common sequence type (ST) was ST95 (n=12), followed by the ST428-, ST23- and ST350-clonal complexes (CCs) (n=8, n=7 and n=6, respectively). The emerging, antimicrobial resistance-associated clones, ST131 and ST648, were represented by five and three isolates, respectively, whereas ST352 and the ST168 CC comprised four isolates each. Phylogroup-B2 isolates showed a greater prevalence of nine virulence genes (P<0.05). One specific clonal group was significantly associated with phylogroup-B2 isolates (P<0.001), and with isolates that induced secondary septicemia (P=0.001). PFGE analysis revealed 12 clusters of genetically related strains (2-12) sampled from unrelated and geographically distant farms, indicating the widespread distribution and recent vertical transmission of particular APEC lineages. Certain lineages showed more diversity, substantiating that long-term, endemic transmission has been maintained. In conclusion, endemic lineages of E. coli that cause salpingitis and peritonitis in broiler breeders, although diverse, tend to be phylogenetically related, and demonstrate conserved virulence genotypes that might be associated with greater pathogenic potential., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The normal gastrointestinal flora of poultry includes Enterococcus faecalis. E. faecalis is also associated with first week mortality of chickens, but it is not clear whether this is due to vertical or horizontal transmission. Aims of the present study were to investigate transmission and genetic diversity of E. faecalis during hatching of broiler chicks. When hatching started, 15% of the chicks were colonized with E. faecalis. This colonization was interpreted as vertical transmission and was higher than previously reported. Transmission of E. faecalis from parents older than 42 weeks was five times greater than transmission of E. faecalis from younger parents. Seventy percent of broiler chicks were colonized with E. faecalis within 24 h after hatch started, which was interpreted as horizontal transmission. Twenty-one sequence types (STs) were demonstrated among 322 isolates of E. faecalis obtained from newly hatched chicks representing 11 different broiler parent flocks. Furthermore, three STs (ST59, ST82, ST174) made up 50.6% of the isolates, indicating that these STs have adapted successfully to the avian niche. All STs, except those novel to this study, have previously been associated with lesions in poultry, underlining the importance of controlling these particular STs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gallibacterium anatis has been suggested to have a causal role in the salpingitis/peritonitis complex in chickens, beside its isolation from the respiratory tract. As G. anatis strains from different flocks were compared by MALDI-TOF MS proteomic phenotyping it could be demonstrated that in most flocks one clonal lineage was present. This finding is also reflected by data achieved when isolates from different organs within a bird generally belong to the same clonal lineage. In addition, it was also confirmed by two independent experiments, as well as, two MALDI instruments. Altogether, proteomic phenotyping indicates that the nature of a chicken flock may play a certain role in particular clone type selection of G. anatis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Avian pathogenic Escherichia coli (APEC) is associated with several types of extraintestinal infections, collectively known as colibacillosis. A heterogeneous population structure has hindered development of vaccines protective against all APEC. Recently, however, the existence of different APEC subpathotypes have been suggested, which are defined by specific disease syndromes and associated virulence genes. A collection of 14 APEC isolates representing clonal outbreaks of salpingitis accompanied by peritonitis and sepsis were characterized in the present study. All the strains carried large plasmids and the aim of the study was to investigate the similarity of these by sequencing, annotating and comparative analysis to identify potential vaccine targets. In addition, a comparison with gene content of human extraintestinal E. coli (ExPEC) subtypes was conducted. Results obtained demonstrated highly similar plasmid contents of the 14 APEC strains, despite the diversity of their chromosomal background. All 14 APEC carried the colicin V operon and numerous virulence genes. These included iss, traT, hlyF, eitABC, ompT, iroBCDEN, sitABCD, iutA and lucABCD. Several of these are shared with human ExPEC, implicating a possible zoonotic potential. Despite a diverse chromosomal background, it was concluded that the plasmid content of virulence genes are highly similar for the investigated APEC subpathotype. Based on their frequency, protein uniformity and subcellular localization iroN, iutA, iss, traT, ompT and etsC are suggested as vaccine-candidates. Experimental studies are, however, necessary to determine the protective potential of the candidates against the APEC subpathotype characterized by salpingitis, peritonitis and possibly septicaemia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Pasteurella multocida is a widespread respiratory pathogen in pigs associated with atrophic rhinitis and contributing to aggravation of the pulmonary lesions. The aims of the present study were to characterize isolates of P. multocida from porcine bronchopneumonia by pulsed-field gel electrophoresis (PFGE), PCR based capsular typing and multilocus sequence typing (MLST) and to compare clonal complexes outlined with the type of histological lung lesions to investigate if a correlation between clonal lineages and lesions might exist. Isolates of P. multocida were obtained from cases of cranioventrally located porcine bronchopneumonia. All lung lesions were described and classified according to histological lesions. A total of 139 isolates, from lung (n=111), pericardial sac (n=21) and kidney (n=7) of 111 pigs were described using PFGE with ApaI as the restriction enzyme. Furthermore, 20 and 29 isolates were characterized by capsular serotyping and multilocus sequence typing, respectively. PFGE demonstrated 15 different clusters showing 50% or more similarity. All selected isolates were of capsular serotype A and only three main sequence types (ST) were detected among the isolates. Associations were not found between histopathology and clonal complexes of P. multocida. In conclusion, PFGE demonstrated a high diversity of genotypes of P. multocida associated with porcine bronchopneumonia. However, isolates obtained mainly belonged to few STs, indicating that isolates of P. multocida associated with porcine bronchopneumonia originates from a limited number of clonal lineages and therefore might have adapted to porcine hosts. No correlation was demonstrated between genotypes and types of lesions, and extra-pulmonary spreading was only rarely demonstrated., (Copyright © 2011 Elsevier B.V. All rights reserved.)

With the aim to extend the present knowledge on possible systemic spreading of Pasteurella multocida in pigs with bronchopneumonia, the occurrence and associated lesions of P. multocida were described by comparing cultural detection, pathological evaluation and in situ hybridization of P. multocida in lungs, hearts and kidneys from cases of porcine bronchopneumonia. P. multocida was cultivated from the lung lesions in 114 out of a total of 148 cases of porcine bronchopneumonia. Among the 114 cases, P. multocida was also cultivated from the pericardial sacs of 40 pigs and the kidneys of seven pigs. Gross lesions and histological findings included a variety of type and stages of bronchopneumonia in connection to the isolation of P. multocida. Furthermore, chronic fibrous pericarditis, interstitial nephritis and a high proportion of lympho-histocytic nephritis were observed. In situ hybridization identified P. multocida in the majority of the lungs, none of the hearts and in half of the kidneys examined. The results show a possible low rate of systemic spreading of P. multocida from lung lesions in pigs with bronchopneumonia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This study was performed to determine the origin and transmission of beta-lactam- and (fluoro)quinolone-resistant Escherichia coli in healthy, untreated broiler flocks. We focused on the dynamics of bacteria resistant to critically important antimicrobials for public and veterinary health in view of the possible link between antimicrobial resistant bacteria in farm animals and humans. By processing faecal samples collected with the sock method in broiler parent and broiler flocks, E. coli resistant to ampicillin and nalidixic acid were frequently isolated, while resistance to ciprofloxacin was detected at a very low frequency, and resistance to cephalosporins was not detected. Similarly, resistance to ampicillin and nalidixic acid were the only phenotypes detected in a collection of clinical E. coli isolates associated with first-week-mortality in broiler parent chicks. Although antimicrobial resistant E. coli were genetically diverse by means of amplified fragment length polymorphism (AFLP) typing, indistinguishable isolates were present in different flocks, including isolates from broiler parent chicks, broiler parents and broilers. In the absence of apparent selective pressure, the genotypic heterogeneity that we describe is likely the consequence of multiple introductions of antimicrobial resistant bacteria into the production system. The confinement under which broilers are raised limits the possibilities of bacterial transmission among different flocks. Our findings are consistent with vertical transmission of ampicillin- and nalidixic acid-resistant E. coli through the broiler production system. The persistence of antimicrobial resistant E. coli in healthy, untreated chicken flocks emphasises the need of careful evaluation of therapeutic options at any level of the broiler production., (Copyright 2009 Elsevier B.V. All rights reserved.)

Pasteurella multocida is responsible for major animal diseases of economic significance in both developed and developing countries whereas human infections related to this bacterium are infrequent. Significantly, development of a carrier status or latent infections plays a critical role in the epidemiology of these diseases. Aiming at increased knowledge of these infections, we examine potential diagnostic and selected typing systems for investigating diseases caused by P. multocida. Detection of P. multocida from clinical specimen by; (i) isolation and identification, (ii) polymerase chain reaction (PCR), iii) specific hybridisation probes, (iv) serological tests and (v) other alternative methods is critically evaluated. These detection systems provide a wide spectrum of options for rapid diagnosis and for detecting and understanding of latent infections in herd/flock health control programmes, though PCR methods for detecting P. multocida in clinical specimen appear increasingly preferred. For establishing the clonality of outbreak strains, we select to discuss macromolecular profiling, serotyping, biotyping, restriction enzyme analysis, ribotyping and multiplex PCR typing. Although P. multocida infections can be rapidly diagnosed with molecular and serological tests, isolation and accurate species identification are central to epidemiological tracing of outbreak strains. Our review brings together comprehensive and essential information that may be adapted for confirming diagnosis and determining the molecular epidemiology of diseases associated with P. multocida.

The epidemiology of an enrofloxacin-resistant Escherichia coli clone was investigated during two separate outbreaks of colibacillosis in the Danish broiler production. In total five flocks were reported affected by the outbreaks. Recorded first-week mortalities were in the range of 1.7-12.7%. The clone was first isolated from dead broilers and subsequently demonstrated in samples from associated hatchers and the parent flock with its embryonated eggs, suggesting a vertical transmission from the parents. The second outbreak involved two broiler flocks unrelated to the affected flocks from the first outbreak. However, the clone could not be demonstrated in the associated parent flock. Furthermore, samplings from grand-parent flocks were negative for the outbreak clone. The clonality was evaluated by plasmid profiling and pulsed-field gel electrophoresis. None of the recognized virulence factors were demonstrated in the outbreak clone by microarray and PCR assay. The molecular background for the fluoroquinolone-resistance was investigated and point mutations in gyrA and parC leading to amino-acid substitutions in quinolone-resistance determining regions of GyrA and ParC were demonstrated. Vertical transmission of enrofloxacin-resistant E. coli from healthy parents resulting in high first-week mortality in the offspring illustrates the potential of the emergence and spreading of fluoroquinolone-resistant bacteria in animal husbandry, even though the use of fluoroquinolones is restricted.

Strains of [Actinobacillus] rossii, [Pasteurella] mairii and [Pasteurella] aerogenes can be isolated from abortion in swine. The RTX toxin Pax has previously been found only in those [P.] aerogenes strains isolated from abortion. Nothing is known about RTX toxins in field isolates of the other two species. To gain insight into the distribution of selected RTX toxin genes and their association with abortion, PCR screening for the pax, apxII and apxIII operons on 21 [A.] rossii and seven [P.] mairii isolates was done. Since species can be phenotypically misidentified, the study was backed up by a phylogenetic analysis of all strains based on 16S rRNA, rpoB and infB genes. The pax gene was detected in all [P.] mairii but not in [A.] rossii strains. No apx genes were found in [P.] mairii but different gene combinations for apx were detected in [A.] rossii strains. Most of these strains were positive for apxIII, either alone or in combination with apxII. Whereas pax was found to be associated to strains from abortion no such indication could be found with apx in [A.] rossii strains. Phylogenetically [A.] rossii strains formed a heterogeneous cluster separated from Actinobacillus sensu stricto. [P.] mairii strains clustered with [P.] aerogenes but forming a separate branch. The fact that [P.] aerogenes, [P.] mairii and [A.] rossii can phylogenetically clearly be identified and might contain distinct RTX toxin genes allows their proper diagnosis and will further help to investigate their role as pathogens.

The taxonomy of the members of the genus Actinobacillus associated with animals has been reviewed with focus on classification and identification including molecular based characterization, typing and identification. Out of the 22 species or species like taxa reported as Actinobacillus, 19 are associated with animals. When classified on the basis of 16S rRNA sequence based phylogenetic analysis, DNA-DNA hybridizations and phenotypic analysis, Actinobacillus sensu stricto is restricted to include A. lignieresii, A. pleuropneumoniae, A. equuli subsp. equuli, A. equuli subsp. haemolyticus (taxon 11 of Bisgaard), A. hominis, A. suis, A. ureae, A. arthritidis (taxon 9 of Bisgaard), Actinobacillus genomospecies 1 and 2 and the taxa 8 and 26 of Bisgaard. The remaining 11 species of Actinobacillus are unrelated to A. sensu stricto and should consequently be grouped with other genera or be renamed as new genera depending on new data. Identification of members of Actinobacillus at species level is possible through phenotypic characterization combined with information on host of isolation. PCR tests are available for specific detection of A. pleuropneumoniae. Only A. pleuropneumoniae is presently considered as a primary pathogen. Based on different types of RTX genes it is possible to PCR type A. pleuropneumoniae to serotype level. PCR might also be used for the specific detection of A. equuli subsp. haemolyticus. Epidemiological investigations and surveillance have so far included serotyping, multilocus enzyme electrophoresis (MLEE), ribotyping and restriction fragment length profiling.