G Charlier

Role of Tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

ABSTRACT Attaching and effacing (A/E) rabbit enteropathogenicEscherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for theeae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tirmutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for theeae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops).

The ability of a high virulence strain (AJS 1) and a low virulence strain (AJS 4) of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of black mollies (Poecilia sphenops) was investigated. For that purpose, two groups of 25 black mollies each were immersed in a bath containing 10(6) CFU/ml of F. columnare AJS 1 or AJS 4. At regular intervals from 1 to 12 h after the contact infection, fish were sacrificed and gills, skin, spleen and heart were sampled for bacteriology. Samples of the gills were taken for immunohistochemical and electron microscopic examination. Bacteriological examination proved that the number of gill-associated F. columnare was higher for AJS 1 than for AJS 4. Strain AJS 1 was isolated from the heart and spleen of 6 and 1 of the 16 examined animals, respectively. Strain AJS 4 was not isolated from the internal organs of any fish. When examined immunohistochemically, strain AJS 1 was found closely associated with gill epithelium whereas this was not the case for strain AJS 4. The adherence of bacteria to the gill tissue challenged with the virulent strain AJS 1 was also clearly demonstrated using scanning and transmission electron microscopy. These results indicate that adhesion of F. columnare to the gill tissue constitutes an important step in pathogenesis.

Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells

Actinobacillus pleuropneumoniae biovar 1 serotypes 2, 5a, 9 and 10 strains were tested for their ability to adhere to alveolar epithelial cells in culture. For the serotypes 5a, 9 and 10 strains, optimal adherence was observed after growth of bacterial cells in a NAD-restricted medium (0.001% NAD). This condition was also associated with the expression of a 55 kDa outer membrane protein (OMP) and of fimbriae. For the serotype 2 strain, adherence and expression of fimbriae and a 55 kDa OMP was less influenced by the growth conditions. The N-terminal amino acid sequence of the 55 kDa OMP had no homology with any known sequence, suggesting that it is an as yet unknown protein. Adherence capabilities were significantly reduced following treatment of the bacteria with proteolytic enzymes or heat. These findings suggest that proteins are involved in adhesion. The hydrophobic bond-breaking agent tetramethylurea was unable to inhibit the adherence of A. pleuropneumoniae to alveolar epithelial cells. Treatment of the bacteria with sodium metaperiodate resulted in lower adhesion scores for the serotypes 2 and 9 strains but the inhibition of adhesion was clearly lower than after treatment with proteolytic enzymes. This indicates that, besides proteins, carbohydrates might also be involved in adhesion of A. pleuropneumoniae to alveolar epithelial cells. The finding that inhibition of adhesion was very high when bacteria were treated with a combination of sodium metaperiodate and pronase also suggests that more than one adhesin is involved.

The long-term cytoskeletal rearrangement induced by rabbit enteropathogenic Escherichia coli is Esp dependent but intimin independent

Attaching and effacing rabbit enteropathogenic Escherichia coli (REPEC) of the O103 serogroup adhere diffusely on HeLa cells and trigger a slow progressive cytopathic effect (CPE) characterized by the recruitment of vinculin and the assembly of actin stress fibres. In contrast to REPEC O103, the reference human EPEC strain E2348/69 is unable to trigger the CPE. In this study, we have shown first that the fimbrial adhesin AF/R2, which mediates the diffuse adhesion of REPEC O103, was not sufficient to induce the CPE capability upon E2348/69. Non‐polar mutants of REPEC O103 for espA, espB, espD and eae were then constructed. The four mutants were unable to induce attaching and effacing lesions in the rabbit ileal loop model. The esp mutants were no longer able to induce the CPE, whereas the eae mutant still induced the CPE. Each espA, ‐B, ‐D mutant could be fully complemented in trans by the corresponding cloned esp genes from both the parental strain and the CPE‐negative E2348/69 strain, indicating that no single esp encodes the information needed to confer the CPE phenotype. In conclusion, the CPE is the first example of an Esp‐dependent but Eae (intimin)‐independent alteration of the host cell cytoskeleton by certain EPEC strains.

Ultrastructure of surface components of Streptococcus gallolyticus (S. bovis) strains of differing virulence isolated from pigeons

Virulence of Streptococcus gallolyticus (S. bovis) strains isolated from pigeons is associated with the presence of the extracellular proteins A, T1, T2 and T3. Based on the presence or absence of these proteins, six supernatant-phenotypes are distinguished. Experimental infection studies have indicated that strains belonging to the A-T1, A+T1, A+T2 and A+T3 groups are highly virulent for pigeons, strains belonging to the A-T3 groups are moderately virulent and A-T2 strains are of low virulence. In this study the surface structure of 15 pigeon S. gallolyticus strains representing high, moderate and low virulence supernatant-phenotypes was examined by electron microscopy. The presence of capsular material was determined by transmission electron microscopy after polycationic ferritin labelling and immunostabilization. Capsules from cells labelled with polycationic ferritin were usually thicker than those from cells exposed to antiserum. The capsule of the virulent strains had a regular, continuous appearance whilst irregularity of the capsule was a characteristic of the low virulence A-T2 strains. Negative staining revealed the presence of fimbriae in all strains belonging to the high virulence A-T1, A+T1, A+T2 and A+T3 supernatant groups and in one strain of the moderately virulent A-T3 group. The fimbriae were thin, flexible structures with a diameter of approximately 3-4 nm and a length of up to 700 nm. Fimbriae as described above were absent in two other A-T3 strains examined and in the low virulence A-T2 strains. Results from this study indicate that morphological differences in surface structure exist among virulent and low virulence pigeon S. gallolyticus strains, and that the capsule and/or fimbriae are possibly involved in virulence.

Early in vivo interactions of Actinobacillus pleuropneumoniae with tonsils of pigs.

Twenty gnotobiotic piglets were inoculated with 5 x 10(8) colony forming units of an Actinobacillus pleuropneumoniae biotype 1-serotype 9 strain onto their tonsils. Five other piglets (controls) were inoculated with phosphate-buffered saline solution. Pigs were euthanized at 30 min, 90 min, 180 min, 6 h, 9 h, 12 h or 24 h after inoculation. At necropsy, samples were taken from the tonsils for bacteriological, histological, immuno-histochemical and electron microscopical examination. A. pleuropneumoniae was isolated from tonsils of all the infected pigs, but not from tonsils of the control pigs. Early after inoculation bacteria were mainly associated with the stratified squamous epithelium and detached epithelial cells. Vacuolization and desquamation of the epithelium was observed and many transmigrating neutrophils were present. At later times after inoculation, bacteria were found closely associated with the crypt-walls and with detached cells present in the crypts. A strong neutrophil migration was observed mainly in the deeper parts of the crypts. It is concluded that attachment of A. pleuropneumoniae to tonsillar epithelial cells probably constitutes a first step in establishing bacteria at this body site.

Clinical signs, reproduction of attaching/effacing lesions, and enterocyte invasion after oral inoculation of an O118 enterohaemorrhagic Escherichia coli in neonatal calves.

Abstract Attaching and effacing (AE) lesions are produced among others by enteropathogenic Escherichia coli and enterohaemorrhagic E. coli (EHEC), which differs from the former by the production of cytotoxins active on various cell cultures, the verocytotoxins, or shigacytotoxins. EHEC are associated with diarrhoea and dysentery in humans and in ruminants, mainly calves from two to eight weeks of age. Clinical signs and/or lesions have been reproduced experimentally with EHEC strains belonging to serotypes O5:K4/Nm, O26:K-:H11, O111:Nm, and O157:H7 which are isolated from cattle and/or humans. The purpose of this work was to develop an experimental model of infection in newborn calves with a bovine EHEC strain isolated from a calf which of died of diarrhoea, and belonging to the O118:H16 serotype, which is also common to both cattle and humans. The bovine O118:H16 EHEC strain was able to colonize the gut of three newborn calves, and to induce diarrhoea twenty-four hours after challenge and to produce AE lesions in the small and/or large intestines. AE lesions were detected microscopically and ultrastructurally in the small intestine of one calf and in the whole intestinal track of two calves. Internalization of bacteria and also of pedestal-bacteria complex inside of the enterocyte was observed in two of the three calves. The significance of this stage is unknown but may be related to the invasion of the calf by the bacteria. The challenge strain was isolated from the mesenteric lymph nodes of the same two calves but not from other organs or from heart blood. No blood was observed in the faeces of any of the three calves, nor were any lesions in the internal organs, which may have been related to the production of a verotoxin whose role is still unknown in cattle.

Intracellular survival and multiplication of virulent and less virulent strains of Streptococcus bovis in pigeon macrophages

The intracellular fate of pigeon S. bovis strains ingested by macrophages was studied in vivo and in vitro. During in vivo experiments, histological and electron microscopical examinations demonstrated numerous cocci, which appeared to be actively multiplying, within splenic macrophages of pigeons experimentally inoculated with a highly virulent S. bovis serotype 1 strain. In pigeons inoculated with a low virulence serotype 3 strain, intracellular cocci were only occasionally observed. For in vitro experiments, pigeon peritoneal macrophages were inoculated with a S. bovis serotype 1 or serotype 3 strain and incubated. Following an initial decrease, an increase in the number of intracellular bacteria was observed in tests performed with the S. bovis serotype 1 strain, demonstrating intracellular multiplication. Macrophages in these experiments had all died after 7 h of incubation, possibly indicating that the intracellular replication of S. bovis resulted in the release of substances toxic for macrophages. In experiments performed with the S. bovis serotype 3 strain, the number of intracellular bacteria continuously decreased, reflecting killing of organisms. Significant changes in the number of adhering macrophages in S. bovis serotype 3 inoculated cultures were not observed. These results indicate S. bovis in pigeons is a facultative intracellular bacterium and intracellular multiplication may be involved in virulence.

An epidemic of fatal hepatic necrosis of viral origin in racing pigeons (Columba livia)

In October 1992, a new disease characterised by acute death in racing pigeons of all ages was noted among the Belgian pigeon population. Clinical symptoms were minimal since affected birds usually died within 24 h. Mortality in pigeon lofts usually amounted to 30%. The most typical necropsy finding was a yellow, pale and swollen liver. Histological and cytological examinations revealed extensive focal or diffuse hepatic necrosis and the presence of eosinophilic or amphophilic nuclear inclusion bodies. This hepatic necrosis of viral origin was diagnosed in 151 (15%) of 988 submissions of pigeons at our Institute in 1993 and 1994. Oral and intramuscular inoculation of pigeons with a bacteria-free homogenate prepared from the liver of pigeons that died from the disease did not result in clinical signs. However, when similar inoculations were done in pigeons previously treated orally with dexamethasone, pigeons died very suddenly within 4-5 days of inoculation. Necropsy findings in experimentally infected pigeons were identical to those observed in naturally occurring cases. Electron microscopic examination of the liver of inoculated pigeons showed the presence of numerous adenovirus-like particles inside nuclei of hepatocytes. The virus could not be cultured in the allantoic cavity or on the chorioallantoic membrane of embryonated chicken eggs. Clinical signs and lesions described here are clearly different from adenovirus enteritis in young pigeons abundantly described since 1976.

Respiratory adenovirus-like infection in a rose-ringed parakeet (Psittacula krameri).

Intranuclear inclusions were observed under light microscopy in the bronchial epithelial cells of a recently purchased female rose-ringed parakeet that died of chlamydiosis. Transmission electron microscopy revealed the presence of numerous particles of adenovirus morphology. A latent adenovirus infection may have become more severe following chlamydiosis and the stress of handling.