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

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.

Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide.

The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro. Purified monocytes were inoculated with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M. hyorhinis, inoculation with C. psittaci did not induce NO production in these phagocytes. The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy. In contrast, upon co-inoculation of the monocytes with C. psittaci and M. hyorhinis, a reduced replication rate of C. psittaci was observed. N(G)-monomethyl-L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M. hyorhinis co-inoculated turkey monocytes. These results imply two considerations. First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection. Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo. Moreover, it was shown that avian M. gallisepticum strains were also able to induce NO in turkey monocytes. Considering the high prevalence of both C. psittaci and Mycoplasma sp. in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys.

Complete development of Cryptosporidium parvum in MDBK cells.

Sporozoites of Cryptosporidium parvum excysted in vitro from bovine oocysts were incubated with monolayers of Madin-Darby bovine kidney cells. The extent of parasite colonisation was monitored by light microscopy and immunofluorescence. Electron microscopy confirmed the complete development and replication of C. parvum within Madin-Darby bovine kidney cells.

Bovine attaching and effacing Escherichia coli possess a pathogenesis island related to the LEE of the human enteropathogenic Escherichia coli strain E2348/69.

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69.

Immunological characteristics of human ferritins; consequences for human serum ferritin determination.

Standard curves established with human spleen, liver, placenta and heart ferritins for eight commercial radioimmunological procedures show that liver and spleen ferritins present almost identical responses, whereas three times as much placenta ferritin and six times as much heart ferritin were required to give the same response as liver and spleen ferritins. There are great variations between the kits in the estimation of the ferritin level of a control serum.

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.

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.

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.

The hyaloid vascular system of the pig. A light and scanning electron microscopic study.

The hyaloid vascular system of the pig was studied from 4 weeks of gestation until 2 weeks after birth by means of semithin sections and vascular corrosion casts. The vascular tunic of the lens is supplied by the posterior lens branches of the hyaloid artery (at the posterior lens pole), by the intermediate lens branches of the proper hyaloid arteries (at the lens equator) and by the anterior lens branches of the radial iridial arteries (at the anterior lens pole). Venous drainage takes place via the venous lens branches which leave the lens anteriorly and drain into the radial iridial veins. Regression of the vascular tunic of the lens occurs during the second half of fetal life and is nearly completed in the first postnatal days. The involution first affects the proper hyaloid arteries and their intermediate lens branches. Subsequently, the posterior lens branches regress, whereas the anterior lens branches in the pupillary membrane disappear in the perinatal period only.

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.

Isolation of orthoreoviruses from psittacine birds.

Orthoreoviridae were regularly isolated from imported psittacine birds in the absence of other pathogens or in combination with salmonella. These viruses grew in embryonated eggs, in chicken embryo fibroblasts and in hepatic cell cultures. The viral isolates were classified as orthoreoviridae on the basis of their morphological and physico-chemical properties.

Pathological findings in two fin whales (Balaenoptera physalus) with evidence of morbillivirus infection.

Two immature female fin whales stranded on the Belgian and French coastlines, were examined post mortem. The main gross findings were massive parasitic infestation, associated with a large thrombus in one whale, and severe emaciation. Microscopical investigations revealed multinucleated syncytia with large intranuclear inclusion bodies in various tissues, and positive immunolabelling for morbillivirus antigens. Other evidence of morbillivirus infection was provided by the demonstration of specific viral structures in syncytia and in cell cultures, and the detection of neutralizing antibodies to canine distemper virus. To the authors>> knowledge, this is the first firm report of morbillivirus infection in baleen whales.

Adenovirus enteritis in pigs.

Experimental transmissions were done with adenovirus strain 6618 in hysterectomy-produced colostrum-deprived pigs. After an incubation period of 3 to 4 days, all inoculated animals had diarrhea. Histopathologically, many intranuclear inclusion bodies were present on short villi of the terminal parts of jejunum and ileum. With electron microscopy, the inclusion bodies were observed to contain numerous adenovirus particles. Immunoperoxidase-positive cells were seen on short villi of the terminal parts of jejunum and ileum. Adenovirus particles also were detected by negative staining of intestinal contents. In 1 pig (naturally occurring infection), adenovirus enteritis was studied by the aforementioned techniques. Similar intestinal lesions as described in the experimental pigs were observed.

Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli.

The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated. An enterotoxin-negative derivative (25KHO9st) was used for further studies. Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein. Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient. The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st. The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes. Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.

A morphometric and functional study of the toxicity of atmospheric ammonia in the extrathoracic airways in pigs.

The effects of atmospheric ammonia (NH3) on the nasal and tracheal mucosa of pigs were investigated by morphometric and functional methods. Pigs were exposed to four concentrations of NH3 [5 (control), 25, 50 and 100 ppm] for 6 days in a specially designed air-pollutant exposure chamber. Samples were taken from the turbinates and the trachea, and the respiratory mucosa was examined by light and scanning electron microscopy. Dose-response curves to carbachol and isoproterenol were constructed using isolated strips of tracheal smooth muscle, with or without epithelium. In pigs exposed to ammonia, considerable mucosal injuries were observed in the turbinates but not in the trachea. The number of neutrophils in the epithelial layer and in the lamina propria, and epithelial hyperplasia were closely and significantly correlated with the concentrations of ammonia (r = 0.894, p < 0.001; r = 0.727, P < 0.001; and r = 0.818, p < 0.001, respectively). Except for the lamina propria, all these changes were significant (p < 0.05) at ammonia concentrations as low as 25 ppm. The percentage of the surface of the turbinate mucosa that was ciliated tended to decrease with increasing ammonia concentration (r = 0.439, p < 0.082). Ammonia induced smooth-muscle hyperresponsiveness to carbachol with a close linear correlation between individual values of the carbachol-induced maximal effect and the NH3 concentrations (r = 0.526, p < 0.003). While mechanical destruction of the epithelium induced an increase in Emax in the control group, no difference was observed between the intact and denuded strips from animals exposed to ammonia. The response to isoproterenol was not influenced by ammonia. It was concluded that quantitative histological analysis of the inflammatory infiltration and epithelial hyperplasia in the turbinates is a useful tool for quantifying the effects of atmospheric pollutants in pigs; a 6-day exposure to ammonia induces nasal irritation and functional disturbances of the tracheal smooth-muscle contractions at concentrations as low as 25 ppm.

Epidemiological study of enteric viruses in broiler chickens: comparison of tissue culture and direct electron microscopy.

The intestinal and caecal contents of chickens from 102 broiler flocks affected by enteric and associated problems were analysed. The second week of life was found to be the most important in the onset of clinical signs of malabsorption shown by the presence of uneven flocks, growth retardation and enteritis problems, but one-week-old flocks frequently presented similar problems. Some cases of feather aberration were observed, mainly in flocks of two weeks of age. From the third week, enteric problems were less acute. Viral particles were found in 67% of the samples by examination by electron microscope and in 52% by cell culture isolation. By complementing the two methods the viral recovery was increased to 85% of the samples. Four virus types were identified: reovirus, entero-like virus, rotavirus and adenovirus. Entero-like virus was mainly found in the first two weeks of life, whilst reovirus and rotavirus were principally found from the second week on. Adenovirus was found infrequently but this may have been a reflection of the mean age of the affected flocks which was only 12 days.

Significance of parvoviruses, entero-like viruses and reoviruses in the aetiology of the chicken malabsorption syndrome.

Specific-pathogen-free White Leghorn chickens and commercial broilers were inoculated orally at 1 day of age with different intestinal preparations containing a chicken parvovirus, an entero-like virus associated with a reovirus from field materials, or the entero-like viruses and reovirus alone. Despite viral multiplication in inoculated birds, no clinical signs or growth retardation were observed in SPF and broiler chickens infected with the reo or parvoviruses. Abnormal faeces and reduction in weight gains were observed after infection with the field materials and the entero-like viruses. Some easily sedimentable particles could be involved with the entero-like virus in the aetiology of runting syndrome. Proventriculitis was present in chickens inoculated with one of the field materials and with the entero-like virus isolated from that material. Specific-pathogen-free White Leghorn chickens were as susceptible as commercial broiler chickens to weight gain depression after oral inoculation with crude homogenates at 1 day of age.