Spontaneous antibiotic resistance mutation associated pleiotropic changes in Escherichia coli O157:H7

Besides the well-known O157:H7 clone causing enterohaemorrhagic colitis and haemolytic uraemic syndrome in Europe, Japan and North America, the number of Escherichia coli isolates with non-motile (NM) phenotype has considerably increased. We supposed that spontaneous antibiotic resistance mutation could cause this phenotypic change. To model our hypothesis we isolated rifampicin--(Rif) and ampicillin--(Amp) resistant mutants from E. coli O157:H7 prototype strains 7785 and EDL933. Among Rifr mutants we could isolate strains with no or reduced motility, while the Ampr mutants became hypermotile. The biochemical profile of the mutants had not changed but phage sensitivity and generation time of the mutants were altered. Among the representative strains we did not find polymorphism with Southern blot analysis and no polymorphism was found in the fliC gene of the mutants. The described characteristics have proven to be stable. In a mice virulence assay by intravenous infections the virulence of the derivatives was also found to be changed. In summary, we found that the antibiotic-resistant phenotype in E. coli O157:H7 was coexpressed with several other phenotypic changes including motility and virulence. It can be assumed that expression of the involved phenotypes may be under the influence of a common regulatory cascade. Further work is needed to identify the components and mechanism of this regulatory system.     

Analysis of gene cassettes of streptomycin-spectinomycin resistance of Hungarian Salmonella enterica serotype Typhimurium strains

By PCR using the ant(3")-Ia primer pair the aadA gene was detected in 34 streptomycin- and spectinomycin-resistant Salmonella enterica serotype Typhimurium strains. Out of them 12 belonged to DT104 and 22 to non-DT104 phage type. Using different primer combinations it was demonstrated that this gene was integron-associated in all cases: in the DT104 strains it was generally contained by a 1 kb integron while in the majority of the non-DT104 strains by a 2.05 kb (less often by a 1.9 or 1 kb) integron. In the case of integrons carrying multiple cassettes the cassette containing the aadA gene was located closer to the 3' end of the integron. The aadA genes of DT104 and non-DT104 strains were different: in the former group the aadA2 gene, while in the latter group (constituted by strains of five different phages types as well as unclassifiable and untypable strains) the aadA1 gene could be identified. The RH50/RH51 primer pair described by Collis and Hall (1992) proved to be suitable for rapid discrimination between the aadA1 and aadA2 genes on the basis that the RH51 primer bound exclusively to the aadA2 gene.     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

Effect of two thermal regimes on the muscle growth dynamics of sea bass larvae,Dicentrarchus labrax L

Muscle growth was studied in larvae of sea bass, Dicentrarchus labrax L., reared at two temperatures: real ambient temperature ( congruent with 15 degrees C during vitelline phase and increased gradually) and 19 degrees C from fertilization until the end of larval development. Muscle cellularity, body length and body weight were measured. Early temperature influenced larval development and so, pre-larval phase finished earlier at 19 degrees C than at ambient temperature (4 and 6 days, respectively). Temperature also affected muscle growth such that at hatching and at mouth opening hypertrophy of muscle fibres was greater at 19 degrees C (P < 0.05), whereas hyperplasia was similar in both groups. After 25 days, the cross-sectional area of the white muscle was greater at 19 degrees C (P < 0.05), which was mainly associated with a higher proliferation of new white muscle fibres. At this stage the body length was also higher at 19 degrees C. Metamorphosis finished earlier in fish reared at 19 degrees C (52 days) than at natural temperature (82 days). At this developmental stage body length and cross-sectional area of the myotome were similar in both groups. However, muscle cellularity differed between groups. Thus, hypertrophy of muscle fibres was higher in fish reared at ambient temperature (P < 0.05), whereas proliferation of new muscle fibres was higher at 19 degrees C (P > 0.05).     

Histological lesions in vascular tissues of bovine herpes virus type 4-infected rabbits

The gamma-herpes virus bovine herpes virus type 4 (BoHV-4) is distributed worldwide in cattle populations with unknown pathogenicity. Bovine endothelial cells were recently shown to be susceptible to BoHV-4 infection in vitro and this virus accelerated the cholesterol-induced atherosclerotic process in rabbits. In this study, the in vivo effect of BoHV-4 on cardiovascular tissue was investigated by intravenous infection of rabbits fed a cholesterol free diet. Inflammatory lesions of vascular tissue in aortic and valvular endothelial cells, and smooth muscle cells were detected by H&E staining, PCR, IF, EM immunohistochemistry, while virus isolation was used to detect virus particles. Acute and chronic vasculitis, signs of chronic endocarditis, with mononuclear cell accumulation and a fresh thrombus was found. Herpes viruses have already been thought to initiate cardio-vascular disorders, now this paper shows that a bovine gamma-herpes virus could also be a causative agent of vascular lesions in mammals fed a normal diet. BoHV-4-infection of rabbits could serve as a useful animal model for research into virus-induced human cardio-vascular diseases.     

Disseminated pythiosis in three horses

Three cases of equine subcutaneous pythiosis with dissemination to the internal organs were investigated. The subcutaneous lesions were observed on the mammary gland, nostrils and limbs of the infected horses. Histopathological analysis of the infected tissues revealed a strong eosinophilic reaction, with macrophages, mast cells and giant cells. Sparsely septated hyphal filaments of 4-6 microm diameter were identified in the center of the eosinophilic areas. Specific fluorescent antibody against Pythium insidiosum confirmed the hyphae in the infected tissues in three examined horses. In one of the three cases, the DNA sequences amplified from the infected subcutaneous tissues and internal organs, revealed that P. insidiosum's 18S SSU rDNA amplicons shared 100% identity with those sequences deposit in GenBank. This is the first report confirming by immunochemical and genetic techniques that P. insidiosum can disseminated from superficial to deep structures.     

Effect of tilmicosin on chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages

OBJECTIVE: To evaluate chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages (AM) exposed to tilmicosin. ANIMALS: 12 healthy calves and 12 healthy pigs. PROCEDURES: Lungs were obtained immediately after euthanasia; AM were collected by means of bronchoalveolar lavage and density gradient centrifugation. Chemotactic activity was evaluated by exposing AM to lipopolysaccharide or macrophage inhibitory peptide during incubation with tilmicosin. Phagocytic activity was evaluated by incubating AM with tilmicosin for 24 hours and then with tilmicosin-resistant Salmonella serotype Typhimurium. Bactericidal activity was evaluated by incubating AM with tilmicosin (0, 10, or 20 microg/ml for bovine AM; 0 or 10 microg/ml or 10 microg/ml but washed free of tilmicosin for porcine AM) and then with Mannheimia haemolytica (bovine AM) or with Actinobacillus pleuropneumoniae or Pasteurella multocida (porcine AM). RESULTS: Tilmicosin had no significant effects on chemotactic or phagocytic activities of bovine or porcine AM. The time-course of bactericidal activity was best described by polynomial equations. Time to cessation of bacterial growth and area under the time versus bacterial number curve were significantly affected by incubation of AM with tilmicosin. CONCLUSIONS AND CLINICAL RELEVANCE: Results show that bactericidal activity of bovine and porcine AM was enhanced by tilmicosin, but not in proportion to the reported ability of AM to concentrate tilmicosin intracellularly. With or without exposure to tilmicosin, the time-course of bactericidal activity of bovine AM against M haemolytica and of porcine AM against A pleuropneumoniae or P multocida was too complex to be reduced to a simple linear equation.     

Use of intraluminal nitinol stents in the treatment of tracheal collapse in a dog

Tracheal collapse is a common problem that is typically observed in middle-aged and older small-breed dogs. It is a structural, obstructive airway disease with a dynamic component that can affect the intra- and extrathoracic portions of the trachea and mainstem bronchi. Many methods of treatment have been suggested, including medical management and provision of extraluminal and intraluminal support. All techniques used to treat intrathoracic and mainstem bronchial collapse have been associated with major complications or limitations. This report describes the implantation of intraluminal nitinol stents to successfully treat intrathoracic as well as extrathoracic tracheal collapse in a dog. The stents are composed of material that has characteristics similar to those of the trachea; nitinol stents may provide a method of supporting intrathoracic tracheal and mainstem bronchial collapse in dogs.     

Reovirus infection in psittacine birds (Psittacus erithacus): morphologic and immunohistochemical study

In this paper we report on an outbreak of reovirus, herpesvirus (Pacheco disease), and/or mycosis infection (Aspergillus spp. and Zygomyces spp.) affecting a batch of young African grey parrots (Psittacus erithacus), with 80% morbidity and 30% mortality. Study material was taken from five birds (four dead and one euthanatized) with a range of clinical symptoms (depression, diarrhea, respiratory symptoms). Diagnosis was confirmed by immunohistochemical detection of avian reovirus, electron microscopy, and virus isolation. Viral antigen of reovirus was detected mainly in large mononuclear cells in the bursa of Fabricius and the spleen, pancreas epithelial cells, and circulating cells; lymphoid organs displayed the largest number of immunopositive cells and severe lymphocyte depletion. Bacteriologic study was negative. Reovirus infection was common in all birds studied, whereas Pacheco disease and mycosis were found in only some, suggesting that reovirus could be the initial cause triggering the outbreak and facilitating infection by other agents and their swift spread through the batch.