Clonality and virulence traits of Escherichia coli associated with haemorrhagic septicaemia in turkeys.

Fifty-five clinical isolates of avian pathogenic Escherichia coli (APEC) from seven outbreaks of acute haemorrhagic septicaemia in turkeys were characterized by serotyping, plasmid profiling including restriction analysis with HindIII, ribotyping with EcoRI and HindIII, multilocus sequence typing (MLST) and virulence profiling. A clonal relationship was demonstrated for each outbreak according to serotype, plasmid profiling, ribotyping, and MLST. In addition, isolates demonstrated highly similar virulence profiles, as all isolates were positive for F11 pili and possessed genes encoding aerobactin (iucD), increased serum survival (iss), temperature-sensitive haemagglutinin (tsh) and colicin V plasmid operon genes (cva/cvi). However, only 20% of the isolates produced colicin V and 42% exhibited serum resistance. All strains with O group O111 and a single O18ac strain (demonstrating non-clonal DNA profiles) were positive for enteroaggregative heat-stabile toxin (EAST1), while isolates of a single outbreak all possessed the enteroaggregative toxin gene (astA). All isolates were negative for genes encoding verocytotoxins (vtx/stx), iron-repressible protein (irp2), P-fimbria (papC), invasion plasmid antigen (ipaH), attaching and effacing gene (eae), enterohaemolysin (ehxA), and enterotoxins LT, STIa (ST(p)) and STIb (ST(h)). In conclusion, highly similar virulence profiles were demonstrated for isolates of E. coli associated with a single well-defined lesion type of colibacillosis in turkeys; acute haemorrhagic septicaemia. The isolates obtained, however, demonstrated a different phylogenetic background, underlining the importance of using well-defined strain collections for characterization of APEC pathotypes.

Association of Streptococcus pluranimalium with valvular endocarditis and septicaemia in adult broiler parents.

The genus Streptococcus consists of more than 60 species, but only Streptococcus equi subspecies zooepidemicus, Streptococcus gallolyticus ssp. gallolyticus, Streptococcus gallinaceus, Streptococcus dysgalactiae, Streptococcus mutans and Streptococcus suis have been isolated from poultry. During investigations of the aetiology of increased mortality in broiler parent stock at the end of production, pure cultures of streptococcal-like organisms that could not be classified among these six species were obtained from 24 cases of septicaemia or valvular endocarditis and septicaemia. Phenotypic characterization using the API20 STREP kit identified the isolates as Aerococcus viridans (10), Aerococcus urinae (2), Leuconostoc species (4), Streptococcus salivarius (2), Streptococcus bovis II 3 (1), Enterococcus avium (3), Enterococcus faecium (1) or Gemella morbillorum (1). However, this identification was misleading as subsequent genetic investigations using pulse field gel electrophoresis and sequencing of 16S rRNA genes showed that 19 isolates were classified as Streptococcus pluranimalium, while the remaining isolates were E. avium (3), E. faecium (1) or Lactobacillus species (1). Misidentification by API20 STREP was related to the database provided by the manufacturer, as the phenotypic characteristics could identify these organisms as S. pluranimalium. The isolates of S. pluranimalium belonged to at least three different clones as determined by pulsed field gel electrophoresis of SmaI-digested genomic DNA. The capacity that these isolates had to colonize the valvular endothelium was suggested by the occurrence of valvular endocarditis in 12 of 19 cases. Demonstration of the same clone in all four houses on a farm suggested the pathogenic potential of this organism.

Characterization of Enterococcus hirae outbreaks in broiler flocks demonstrating increased mortality because of septicemia and endocarditis and/or altered production parameters.

In Denmark, increased problems associated with streptococci and enterococci have been observed in broilers and broiler parent flocks, resulting in increased mortalities, uneven flocks, and subsequent downgrading and increased condemnations. Postmortem lesions associated with recent outbreaks due to Enterococcus hirae have been accompanied or dominated by septicemia and endocarditis. As a result of infection at an early age and relatively low mortality rates, outbreaks are not always clearly defined and may go unnoticed or may be attributed to poor chick quality. For the same reasons, the pathogenesis and epidemiology of observed outbreaks has only remained speculative. Four separate outbreaks associated with E. hirae infections in broiler flocks occurring between 1998 and 2002 have been investigated. Two of the outbreaks indicated evidence of two separate clones, with 89% and 79% of isolates involved in the individual outbreaks belonging to a single pulsed-field gel electrophoresis (PFGE) profile, respectively. Another outbreak (outbreak 4) demonstrated clear clonality, with all isolates demonstrating affinity to one of two PFGE profiles that differed by only two bands. However, all three outbreaks demonstrated a different clone. The remaining outbreak was nondonal, with isolates distributed between six separate PFGE profiles. One of the outbreaks (outbreak 4) was descended from a parent broiler flock previously associated with an outbreak of Streptococcus gallinaceus; the flock also exhibited septicemia and endocarditis. Initial indications suggested the possibility of vertical transmission of S. gallinaceus to the current broiler flock, causing infection. By extended phenotypic characterization and subsequent genetic characterization, including 16S rRNA sequencing, all strains from the four outbreaks were confirmed as E. hirae. This investigation highlights the problems associated with characterizing enterococci infections in broiler flocks.

Evaluation of treatment and prophylaxis with nitrofurans and comparison with alternative antimicrobial agents in experimental Salmonella enterica Serovar enteritidis infection in chicks.

The ability of the nitrofuran antimicrobial agents furazolidone and furaltadone to prevent, reduce or eliminate Salmonella enterica serovar enteritidis PT4 infection in artificially challenged day-old chicks was evaluated. Treating the birds with the nitrofurans failed to eliminate established infections with either furazolidone-resistant (FzR) or furazolidone-sensitive (FzS) strains. Simultaneous administration of the nitrofurans to day-old chicks challenged with FzS failed to prevent infection but reduced colonization significantly (p<0.05) compared to unmedicated controls. No reduction of colonization occurred with FzR. Challenging birds with FzS and simultaneous dosing with nitrofurans for 1 week, followed by a second week of continued treatment, resulted in an increase in the level of colonization in the second week rather than a decrease. Dosing with the nitrofurans (200 ppm) for 1 week prior to challenge with FzS and continued medication for a further week prevented colonization of the caecum, liver and spleen. However, cessation of dosing at the time of challenge with salmonella resulted in colonization. Chloramphenicol and tetracycline at concentrations of 200 ppm were both independently capable of preventing colonization by salmonella. Sulphadiazine initially reduced colonization but failed to eliminate the infection. Only when furazolidone was combined with chloramphenicol or when sulphadiazine was combined with trimethoprim, and the combined drugs were administered concurrently with the challenge, was colonization prevented.

Pathology, tissue metalloproteinase transcription and haptoglobin responses in mice after experimental challenge with different isolates of Pasteurella multocida obtained from cases of porcine pneumonia.

Pasteurella multocida is a major cause of porcine pneumonia, but the pathogenesis of the disease is poorly defined. The aim of this study was to further understand the host response to infection by use of a mouse model of P. multocida pneumonia. Twenty female mice were divided into four groups (n=5). Three groups were infected with one of three isolates of P. multocida isolated from clinical cases of chronic porcine pneumonia with necrotizing, suppurative and non-suppurative lesions, respectively. The fourth group served as uninfected controls. Mice were killed 24 h postinfection and samples were collected for bacteriology, histopathology and in-situ hybridization for detection of P. multocida. Measurements of expression of genes encoding matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) in lung tissue and quantification of serum haptoglobin concentration were performed. P. multocida was found in the lung and spleen. Lung lesions were characterized by deposition of fibrin in alveoli and bronchioles, perivascular oedema, suppuration and necrosis. The cellular infiltration was mainly of neutrophils. Splenic neutrophilic infiltration was also evident. Minor differences in the severity and nature of lesions were seen according to the isolate of P. multocida used for infection. Intranasal infection of mice can therefore be used to evaluate the host response and lesions caused by P. multocida obtained from porcine pneumonic infections. The inflammatory response in this model is associated with increased tissue expression of genes encoding MMP9, TIMP1 and serum haptoglobin concentration.

Geno- and phenotypic diversity of avian isolates of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis) and associated diagnostic problems.

Recently, strains of Streptococcus bovis were reclassified as Streptococcus gallolyticus. In the present study we describe for the first time an outbreak of S. gallolyticus in a broiler flock. Mortality during the first week was normal (<1%), with a final total mortality at the end of production reaching 4.3%. Specific symptoms were not observed. Postmortem pathology demonstrated enlarged and light spleens and livers accompanied by multifocal irregular necroses surrounded by a hemorrhagic zone. In addition, these birds suffered from arthritis and osteomyelitis. Strains isolated from liver and spleen lesions showed clonality as demonstrated by pulsed-field gel electrophoresis. Compared to strains representing previously derived phylogeny, including the S. bovis-S. equinus complex, the 16S rRNA-derived phylogeny of the strains investigated in this study demonstrated a paraphyletic group (S. gallolyticus) well separated from two monophyletic groups: (i) S. equinus-S. bovis plus S. infantarius and (ii) S. alactolyticus plus S. intestinalis. According to information in GenBank, none of the strains included from the two monophyletic groups have been isolated from birds. Further biochemical analyses, including tannase activity, identified for the first time avian isolates belonging to S. gallolyticus subsp. gallolyticus. However, these investigations also demonstrated a clear heterogeneity with pigeon isolates.

Reproduction of sepsis and endocarditis by experimental infection of chickens with Streptococcus gallinaceus and Enterococcus hirae.

This study describes experimental infections in 4-week-old chickens inoculated intravenously with approximately 10(8) colony-forming units Streptococcus gallinaceus strain CCUG 42692T (C13156) or Enterococcus hirae strain DSM 20160 (C17410). Birds were necropsied following death and obvious clinical signs of disease or were euthanized weekly after infection for up to 4 weeks. At necropsy, lesions included splenomegaly, hepatomegaly, valvular and/or mural endocarditis. Cardiac lesions included focal necrotizing myocarditis and/or yellow-white vegetative valvular endocarditis or greyish proliferations associated with the mitral valves in 35% (6/20) and 79% (19/24) of birds infected with S. gallinaceus and in 20% (4/20) and 55% (12/22) of birds infected with E. hirae via the brachial and jugular veins, respectively. S. gallinaceus was reisolated from heart valves in 45% (9/20) and 75% (18/24) and E. hirae in 35% (7/20) and 73% (16/22) after inoculation via brachial and jugular veins, respectively. Both challenge strains were also isolated from liver, spleen, bone marrow and hock joints. A significant difference between the infections with the two strains was seen only with reisolation of E. hirae from hock joints (P < 0.007). Significant differences were apparent between the two inoculation routes only with E. hirae, where infection via the jugular vein was associated with higher culture positive isolations from the heart (P = 0.029), bone marrow (P = 0.002) and hock joints (P < 0.001) compared with the brachial vein. Birds injected with sterile phosphate-buffered saline were negative for culture of the challenge strains and no lesions were observed in these controls. The results confirm that both S. gallinaceus and E. hirae can cause endocarditis in experimentally infected chickens.

In vitro activity of nitrofuran derivatives on growth and morphology of Salmonella enterica serotype Enteritidis.

AIMS: To evaluate the effect of nitrofuran derivatives furazolidone (Fz) and nitrofurantoin (Nf) on Salmonella enterica serovar Enteritidis PT4 in vitro, with regard to cell growth, morphology and ultrastructure. METHODS AND RESULTS: The effects of Fz on the growth rates of Fz resistant (FzR) and sensitive (FzS) strains were assessed by viable counts. Over 24 h incubation, concentrations of <1 microg ml(-1) of Fz were bacteriostatic to the FzS strain. The FzR strain tolerated concentrations up to 16 microg ml(-1) before cell numbers diminished over the same time period. The effect on the growth rate of the FzS strain after 1 h exposure to supra-inhibitory concentrations of Fz, gave a maximum response at 32X minimum inhibitory concentration (MIC) of 4.5 h. Effects on the ultrastructure of bacterial cells by scanning electron and transmission microscopy, and DNA-specific staining with DAPI of the FzS strain exposed to nitrofurans were studied. Abnormalities such as extensive filamentation with sparse, sporadic nucleotide distribution and evidence of extrusions in the cell envelope in the form of blebs were evident. CONCLUSIONS: Nitrofurans exert their bactericidal effect on Salmonella by inducing extensive structural alteration after exposure at sub- or suprainhibitory concentrations, involving inhibition of cell division because of the activated drug causing an intercalating type of binding in DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the in vitro activity of the nitrofuran derivatives, furazolidone and nitrofurantoin on Salmonella, defining the pharmacodynamics and physical nature of their action as therapeutic agents.

Characterization of streptococci and enterococci associated with septicaemia in broiler parents with a high prevalence of endocarditis.

Increased mortality due to septicaemia, where 29% of the affected birds had developed valvular endocarditis, was observed in a flock of broiler parents affected by myelocytomatosis. Bacterial investigations resulted in isolation of streptococci, the classification of which was unknown according to present taxonomy. Strains were isolated from the liver, spleen, heart and salpinx in clinical cases of septicaemia from the affected flock of Ross broiler parents aged between 26 and 56 weeks. Phenotypic characterization followed by genotypic investigation by ribotyping with HindIII and pulsed-field gel electrophoresis (PFGE) with SmaI demonstrated three ribotypes and six different PFGE profile types, respectively. Ribotype A with variant A1, together with the PFGE profile type represented by the three subtypes Ia, Ib and Ic, dominated the outbreak constituting 85% of the strains investigated, indicating clonality. 16S rRNA sequencing of strains representing this genotype demonstrated the occurrence of a recently described new Streptococcus sp., Streptococcus gallinaceus. Sequence analysis of the other genotypes demonstrated in the outbreak, resulted in identical 16S rRNA sequences to the type strain of Enterococcus faecalis. S. gallinaceus appears to represent a new opportunistic pathogen within the poultry industry.

Application of molecular methods for identification of strains classified as Salmonella enterica serovar 6, 7:-:- by conventional serotyping.

An increased prevalence of Salmonella enterica serovar Tennessee (6, 7: z(29):-) was observed in broiler flocks in Denmark in 1994 and a parallel increase in the prevalence of Salmonella enterica serovar 6, 7:-:- was demonstrated, albeit at a lower level. Plasmid profiling and ribotyping revealed similar genotypes and it was speculated that serovar 6, 7:-:- could represent a non-motile variant of Salmonella Tennessee. Re-testing of the Salmonella 6, 7:-:- isolates demonstrated the presence of flagella through positive motility. All isolates but one demonstrated motility using both tube tests and light microscopy of overnight broth cultures. Molecular characterization indicated that all but two isolates previously classified as Salmonella 6, 7:-:, were isolates of Salmonella Tennessee and Salmonella Infantis, exhibiting reduced motility. Re-serotyping and multiplex polymerase chain reaction analysis for the phase 2 gene fljB demonstrated variants of Salmonella Infantis (6, 7: r: z(49)) expressing the R-phase antigen (Rz(49)) and possessing the gene for normal phase 2 antigen H: 1, 5. One of the two undefined strains demonstrated genotypic identity with a Salmonella Livingstone reference strain. The remaining putative 6, 7:-:- strain could not be identified and was genuinely non-motile. Diagnostic procedures performed initially were thus insufficient to differentiate between the different levels of motility and also resulted in mis-serotyping. As similar observations were made with two of 14 isolates received from a foreign laboratory, this may represent a general diagnostic problem.

Development of an in vivo model for study of intestinal invasion by Salmonella enterica in chickens.

An in vivo loop test model for the investigation of the invasiveness of Salmonella enterica in chickens was developed. Ten jejunal loops were made in 10- to 12-week-old Lohman Brown chickens under isofluoran anaesthesia. Salmonella at 5.0 x 10(7) CFU was inoculated into each loop and left for 2 h, followed by a 1-h incubation with gentamicin in order to kill noninvading bacteria. After euthanasia, Salmonella invasiveness was measured as tissue-associated counts relative to a reference strain. The ability of Salmonella invasion was 1 log(10) CFU higher per 42-mm(2) mucosal tissue in the anterior than in the posterior part of jejunum. A statistically significant (P<0.001) sixfold difference in invasiveness was observed between a wild-type S. enterica serotype Typhimurium strain and the corresponding invH mutant. The model was shown to be able to show small differences in invasive capability and allows for comparison of strains tested in different animals, provided that the same reference strain is present in all animals.

Quantitative comparison of intestinal invasion of zoonotic serotypes of Salmonella enterica in poultry.

The aim of the present study was to compare the invasion of selected zoonotic Salmonella serotypes of poultry in an in vivo chicken intestinal loop model and also in vitro in epithelial cell cultures. Invasion was measured relative to a reference strain, Salmonella Typhimurium 4/74 invH201::TnphoA. Two serotypes demonstrated intracellular log(10) counts that differed significantly from all other serotypes tested: Salmonella Enteritidis PT4 being 1.5 log(10) colony forming units (CFU) (31-fold) higher, and Salmonella Tennessee being 0.7 log(10) CFU (fivefold) lower than the reference strain (P < or = 0.0001). A group of serotypes, which can be vertically transmitted, showed significantly higher intracellular counts (fourfold to eightfold) than the reference strain. The group included S. Typhimurium 4/74, S. Typhimurium DT104 (poultry and porcine isolates), S. Enteritidis PT1, S. Enteritidis PT6, S. Enteritidis PT8, and Salmonella Berta. The serotypes Salmonella Hadar, Salmonella Virchow, S. 4,12:b:-, S. Typhimurium DT41, and Salmonella Infantis, most of which are considered horizontally transmitted, did not show significantly different intracellular counts from the reference strain. Results from the cell culture invasion studies agreed with the in vivo data, with the exception of S. Berta and the poultry isolate of S. Typhimurium DT104.