Preferential transcription of the mutated allele in NPM1 mutated acute myeloid leukaemia.

Nucleophosmin is commonly both over-expressed and mutated in acute myeloid leukemia (AML). NPM1 mutations are always heterozygous. In addition, NPM1 has a number of different splice variants with the major variant encoded by exons 1-9 and 11-12 (NPM1.1). Further variants include NPM1.2 which lacks exons 8 and 10 and NPM1.3 which comprises exons 1-10 (and so lacks the region of sequence mutated in AML). In this study we quantified the expression of these three variants in 108 AML patient samples with and without NPM1 mutations and also assessed the level of expression from the wild-type and mutant alleles in variants NPM1.1 and NPM1.2. The results show that NPM1.1 is the most commonly expressed variant, however transcripts from wild-type and mutated alleles do not occur at equal levels, with a significant bias toward the mutated allele. Considering the involvement of mutant nucleophosmin in the progression and maintenance of AML, a bias towards mutated transcripts could have a significant impact on disease maintenance.

Salmonella and on-farm risk factors in healthy slaughter-age cattle and sheep in eastern Australia.

OBJECTIVE: To examine healthy slaughter-age cattle and sheep on-farm for the excretion of Salmonella serovars in faeces and to identify possible risk factors using a questionnaire. PROCEDURE: The study involved 215 herds and flocks in the four eastern states of Australia, 56 with prior history of salmonellosis. Production systems examined included pasture beef cattle, feedlot beef cattle, dairy cattle, prime lambs and mutton sheep and animals were all at slaughter age. From each herd or flock, 25 animals were sampled and the samples pooled for Salmonella culture. All Salmonella isolated were serotyped and any Salmonella Typhimurium isolates were phage typed. Questionnaires on each production system, prepared in Epi Info 6.04, were designed to identify risk factors associated with Salmonella spp excretion, with separate questionnaires designed for each production system. RESULTS: Salmonellae were identified in all production systems and were more commonly isolated from dairies and beef feedlots than other systems. Statistical analysis revealed that dairy cattle were significantly more likely to shed Salmonella in faeces than pasture beef cattle, mutton sheep and prime lambs (P<0.05). A wide diversity of Salmonella serovars, all of which have been isolated from humans in Australia, was identified in both cattle and sheep. Analysis of the questionnaires showed access to new arrivals was a significant risk factor for Salmonella excretion on dairy properties. For beef feedlots, the presence of large numbers of flies in the feedlot pens or around stored manure were significant risk factors for Salmonella excretion. CONCLUSION: Dairy cattle pose the highest risk of all the slaughter-age animals tested. Some of the identified risk factors can be overcome by improved management practices, especially in relation to hygiene.

Improved diagnosis of virulent ovine footrot using the intA gene.

Footrot is a mixed bacterial infection of the hooves of sheep. The gram-negative anaerobic bacterium Dichelobacter nodosus is the principal causative agent, with different strains causing diseases of different severity, ranging from benign to virulent. In Australia, in the state of New South Wales (NSW), only virulent footrot is subject to regulatory action, including quarantine. However, it is often difficult to distinguish benign footrot from virulent footrot in the initial stages of infection, or under adverse climatic conditions. The gelatin gel test, which measures the thermostability of secreted bacterial proteases, is the laboratory test most widely used in Australia to aid in the differential diagnosis of footrot. The proteases of virulent strains are, in general, more thermostable than the proteases of benign strains. However, there are some false positives in the gelatin gel test, which may lead to unnecessary quarantine procedures. We used Southern blot analysis on 595 isolates of D. nodosus from 124 farms on which sheep had benign or virulent footrot to test for the presence of the intA gene. We found that for D. nodosus strains which are stable in the gelatin gel test, there is a high correlation between the presence of the intA gene and the ability of the strain to cause virulent footrot. We also developed a PCR-based assay for the rapid detection of intA, which can be used to test DNA extracted from colonies grown on plates, or DNA extracted from cotton swabs of culture plates.

Oral associated bacterial infection in horses: studies on the normal anaerobic flora from the pharyngeal tonsillar surface and its association with lower respiratory tract and paraoral infections.

Two hundred and seventy bacterial isolates were obtained from the pharyngeal tonsillar surface of 12 normal horses and 98 obligatory anaerobic bacteria were characterised. Of these, 57 isolates belonging to 7 genera (Peptostreptococcus (1); Eubacterium (9); Clostridium (6); Veillonella (6); Megasphera (1); Bacteroides (28); Fusobacterium (6)) were identified, and 16 of these were identified to species level (P. anaerobius (1); E. fossor (9); C. villosum (1); B. fragilis (1); B. tectum (2); B. heparinolyticus (2)). Three hundred and twenty isolates were obtained from 23 samples from horses with lower respiratory tract (LRT) or paraoral (PO) bacterial infections. Of the 143 bacteria selected for detailed characterisation, obligate anaerobes accounted for 100 isolates, facultative anaerobes for 42 isolates and obligate aerobes for one isolate. Phenotypic characterisation separated 99 of the isolates into 14 genera. Among the obligately anaerobic species, Gram-positive cocci including P. anaerobius comprised 25% of isolates, E. fossor 11% and other Gram-positive rods (excluding Clostridium sp.) 18% of isolates. The Gram-negative rods comprised B. fragilis 5%, B. heparinolyticus 5%, asaccharolytic pigmented Bacteroides 3% and other Bacteroides 13%, while a so-far unnamed species of Fusobacterium (7%), and Gram-negative corroding rods (3%) were isolated. Among the facultatively anaerobic isolates, S. equi subsp. zooepidemicus accounted for 31% of isolates, followed by Pasteurella spp. 19%, Escherichia coli 17%, Actinomyces spp. 9%, Streptococcus spp. 9%. Incidental facultative isolates were Enterococcus spp. 2%, Enterobacter cloaceae 2%, Actinobacillus spp. 2% and Gram-negative corroding rods 5%. On the basis of the similarities (as determined by DNA hybridization data and/or phenotypic characteristics) of some of the bacterial species (e.g. E. fossor and B. heparinolyticus) isolated from both the normal pharyngeal tonsillar surfaces and LRT and PO diseases of horses, it is considered that the most likely source of bacteria involved in these disease processes is flora from the oral cavity.

Chromosomal DNA probes for the identification of Bacteroides tectum and Bacteroides fragilis from the oral cavity of cats.

A dot-blot hybridisation assay using high molecular weight DNA as whole chromosomal probes was used to differentiate Bacteroides tectum from Bacteroides fragilis. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes. The whole chromosomal probes were specific--differentiating B. tectum from B. fragilis and both from a variety of other species (including other members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. However, even at very high stringencies, B. tectum homology groups I, II and III were not distinguishable from one another using either 32P-labelled or DIG-labelled probes. Thus, DIG-labelled whole chromosome probes directed against cellular DNA released directly onto nitrocellulose membranes is considered a useful method for diagnostic veterinary laboratories wishing to identify B. tectum and distinguish it from B. fragilis and other oral anaerobic flora of cats.

Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats.

A dot-blot hybridisation assay using isolated high molecular weight DNA as whole chromosomal probes of the cat pigmented asaccharolytic Bacteroides/Porphyromonas species was used against both purified high molecular weight DNA and DNA released on membranes from whole cells for the identification of B. salivosus and for its differentiation from the other anaerobic species isolated from normal and diseased mouths of cats and horses. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes (Boehringer-Mannheim). The whole chromosomal probes were specific--differentiating B. salivosus from a variety of species (including members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. Likewise, asaccharolytic black pigmented Group 2 strains were distinguishable from all strains tested. However, cat strains of P. gingivalis which show 68-76% DNA-DNA homology with human strain P. gingivalis ATCC 33277T, were not distinguishable from each other using either 32P-labelled or DIG-labelled probes. The minimum amount of pure Bacteroides DNA which could be detected by the 32P-labelled probe was 100-300 pg, while the amount of pure DNA detected by the DIG system was 1-3 mg after room temperature colour development for 1 h and 100-300 pg after 6 h colour development.

Effect of single bouts of moderate and high intensity exercise and training on equine peripheral blood neutrophil function.

The effects of single bouts of moderate (30 to 40 per cent VO(2)max) and high (115 per cent VO(2)max) intensity exercise on equine peripheral blood leucocyte function were evaluated by determining neutrophil phagocytosis and oxidative burst activity before and after treadmill exercise and training. Prior to all exercise tests, the possible effect of diurnal variation was evaluated in samples obtained from four resting horses. Subsequently eight horses underwent moderate and high intensity exercise protocols and then commenced a 17-week training period. High intensity exercise tests were repeated in week 10, after 7 weeks of endurance training, and in week 17, after a further 6 weeks of high intensity training. Time of sampling had a significant effect on neutrophil function for resting, untrained horses. Prior to training, moderate intensity exercise was associated with improved neutrophil phagocytosis and oxidative burst activity. High intensity exercise was associated with transient impairment of these responses. A similar reduction was not demonstrable following high intensity exercise in weeks 10 or 17 of training. Neutrophil function in week 17 was suppressed at all sampling times relative to results obtained in week 10, suggesting that high intensity training may have been associated with a general reduction in neutrophil function.

The effect of high intensity exercise on the functional capacity of equine pulmonary alveolar macrophages and BAL-derived lymphocytes.

The effect of strenuous exercise on the functional capacity of pulmonary alveolar macrophages (PAM) and bronchoalveolar lavage-derived lymphocytes was determined in eight horses prior to and after 7 weeks of training. Strenuous exercise had no effect on the total cell count or the percentage of live cells in bronchoalveolar lavage (BAL) samples prior to or following training. However, training was associated with a significant increase in the total cell count of pre-exercise BAL samples and a significant reduction in the percentage of live cells in post-exercise samples. Strenuous exercise was associated with impaired phagocytosis by PAM after 7 weeks of training but had no effect on similar samples obtained from untrained horses. The oxidative burst activity of PAM was significantly increased following strenuous exercise for both untrained and trained horses. BAL -derived lymphocyte oxidative burst was similarly affected following training. These results suggest that strenuous exercise and training may influence pulmonary immune cell function.

Genetic structure of populations of beta-haemolytic Lancefield group C streptococci from horses and their association with disease.

The genetic structure of beta-haemolytic Lancefield group C streptococci isolated from horses in Australia was examined by multilocus enzyme electrophoresis. The 249 isolates comprised 70 classified phenotypically as Streptococcus equi subspecies equi, 177 classified as S equi subspecies zooepidemicus and two which were unclassifiable. Forty-one electrophoretic types were identified which could be classified into three major clusters, A, B and C. Of the isolates, 178 fell into cluster B (types 4 to 22) and lay within a genetic distance of 0.36. Sixty-nine of the 70 S equi subspecies equi isolates fell into type 12, which suggests that they were members of a single clone, and the isolates from abscesses were significantly more likely to belong to type 12 than those from horses with no clinical signs (P < 0.001). There were no other significant associations between electrophoretic types or clusters and the isolation of the organism from particular sites. These data suggested that S zooepidemicus may be the archetypal species from which the clone designated subspecies equi has been derived. If isolates of the subspecies equi from other geographical regions also prove to be members of electrophoretic type 12, this hypothesis would be strengthened.

Flow cytometric determination of oxidative burst activity of equine peripheral blood and bronchoalveolar lavage-derived leucocytes.

Flow cytometric techniques were developed for the evaluation of oxidative burst activity in equine peripheral blood neutrophils and lymphocytes, as well as bronchoalveolar lavage derived pulmonary alveolar macrophages and lymphocytes. The oxidation of dichlorofluorescin was measured by the increased fluorescence of cells stimulated with phorbol myristate acetate or a variety of other stimulants. Flow cytometry was a suitable method for the evaluation of the intracellular oxidation in all cell populations evaluated. Analysis was rapid and cell separation before analysis was not required. Heterogenous cell populations with differing responsiveness to phorbol myristate acetate stimulated oxidative burst were identified in peripheral blood neutrophil and alveolar macrophage populations. The current study characterizes flow cytometric techniques for the evaluation of oxidative burst activity in equine peripheral blood and bronchoalveolar lavage-derived leucocytes.

The flow cytometric evaluation of phagocytosis by equine peripheral blood neutrophils and pulmonary alveolar macrophages.

Flow cytometry was used to assess the phagocytosis of fluorescent-labelled bacteria by equine peripheral blood neutrophils and pulmonary alveolar macrophages. Cell populations were prepared from venous blood following ammonium chloride lysis and from washed bronchoalveolar lavage derived samples. Discrete clusters of cells, corresponding to different leucocyte groups, were readily identified on the basis of differing light scattering properties and could thus be discriminated, negating the need for prior cell separation. Cells able to associate with fluorescent-labelled bacteria (by attachment to the cell membrane or by internalization within the cell) acquired increased fluorescence and were readily differentiated from cells unable to interact with bacteria. The fluorescence of bacteria attached to the cell surface was quenched by the addition of trypan blue or counterstained by the addition of ethidium bromide to the assay, thus permitting identification of cells which were able to internalize bacteria.

Effects of posture and accumulated airway secretions on tracheal mucociliary transport in the horse.

Tracheal mucociliary clearance was determined in horses by measuring the rostrad transport of the radiopharmaceutical 99mtechnetium-sulphur colloid following deposition on the tracheal epithelium by intratracheal injection. The effects of head position (head elevated to normal standing position vs head lowered) and of accumulated purulent secretions on tracheal mucociliary clearance were evaluated for the first time in the horse. In normal horses tracheal mucociliary clearance was greatly accelerated by lowering the head so that the cranial trachea was lower than the caudal trachea. Horses confined with their heads elevated for 24 hours developed an accumulation of purulent airway secretions (and associated increased numbers of bacteria) in the lower respiratory tract and showed a decrease in tracheal mucociliary clearance when compared with their previously measured rate when the lower airway contained only normal secretions. These findings have implications for management practices where horses are prevented from lowering their heads, such as transportation and cross-tying, which may therefore contribute to lower respiratory tract disease in horses.

Effect of transportation on lower respiratory tract contamination and peripheral blood neutrophil function.

OBJECTIVE: To evaluate the effect of transportation on lower respiratory tract contamination and peripheral blood neutrophil function in horses and to compare results from transported horses with those obtained in earlier experiments from horses confined with heads elevated. DESIGN: A prospective study. PROCEDURE: Six horses were transported by road for 12 h. Clinical and haematological examination, transtracheal aspiration and cell function studies were conducted before and after transportation. Results obtained after transportation were compared to pre-transportation values. RESULTS: After transportation, peripheral blood leucocyte and neutrophil numbers were increased and rectal temperatures were evaluated. Transtracheal aspirates showed an accumulation of purulent respiratory tract secretions with increased numbers of bacteria, particularly beta-haemolytic Streptococcus spp and members of the Pasteurellaceae family. Three horses also had increased numbers of bacteria from the Enterobacteriaceae family relative to corresponding samples from earlier studies. Phagocytosis by peripheral blood neutrophils was significantly reduced, while the oxidative burst activity of peripheral blood leucocytes was either unchanged or enhanced. CLINICAL IMPLICATIONS: Bacterial contamination of the lower respiratory tract occurs as a routine consequence of transportation of horses and is likely to be an important determinant in the development of transport-associated respiratory disease. Inflammatory airway secretions and increased numbers of bacteria were rapidly cleared, without clinical evidence of significant pulmonary disease and without additional treatment, in normal horses that were allowed to lower their heads after transportation. Peripheral blood neutrophilia and a reduction in neutrophil phagocytic function were evident for at least 36 h after transportation, suggesting that horses may require a number of days to recover from the stress of transportation. As the potential role of bacteria from the Enterobacteriaceae family in the development of transport-associated respiratory disease has not been elucidated, horses which develop clinical disease following transportation should undergo thorough bacteriological investigation to ensure appropriate treatment.

Inflammation and increased numbers of bacteria in the lower respiratory tract of horses within 6 to 12 hours of confinement with the head elevated.

Confinement of horses with their heads elevated for periods up to 24 hours was used to evaluate the extent and the effects of bacterial contamination of the equine lower respiratory tract. Significant (P < 0.05) increases in bacterial numbers (up to 10(9) colony forming units/mL in transtracheal aspirate derived samples) occurred within 6 or 12 hours in most horses. Pasteurella/Actinobacillus spp and Streptococcus spp were most commonly isolated. Lowering of the head for 30 minutes every 6 hours to facilitate postural drainage did not prevent multiplication of organisms to levels equivalent to those achieved by horses where the head was elevated for 24 hours. When horses were released from confinement and heads were no longer maintained in an elevated position, clearance of accumulated secretions and bacteria occurred within 8 to 12 hours. Thus, confinement with the head elevated resulted in significant bacterial contamination and multiplication within the lower respiratory tract during a period often encountered in routine management procedures, such as transportation. The clearance of accumulated secretions occurred over a prolonged period after release from such confinement.

Antibiotic prophylaxis of lower respiratory tract contamination in horses confined with head elevation for 24 or 48 hours.

OBJECTIVE: To evaluate the administration of procaine penicillin prior to or during confinement with head elevation as a means of reducing the associated accumulation of inflammatory lower respiratory tract secretions and increased numbers of bacteria within the lower respiratory tract of confined horses. DESIGN AND PROCEDURE: Two experiments were conducted to evaluate the efficacy of different dose rates and dosing frequencies. In experiment A a single low dose (15,000 IU/kg) of procaine penicillin was administered to four horses immediately prior to confinement with head elevation for 48 hours. The systemic leucocyte response, gross and cytologic characteristics of transtracheal aspirate and bacterial numbers in lower respiratory tract samples were compared with corresponding samples from two horses confined with heads elevated but not given penicillin. The efficacy of higher dose rates (20,000 IU/kg and 40,000 IU/kg) given before and during confinement with heads elevated for 24 hours was evaluated in experiment B. RESULTS: Treatment with procaine penicillin had no effect on the systemic leucocyte response or on the accumulation of inflammatory lower respiratory tract secretions at any of the dosing schedules evaluated. The number of bacteria isolated from trans-tracheal samples was reduced at 12 hours for treated horses in experiment A and at 24 hours for experiment B. beta-haemolytic Streptococcus spp were not isolated from treated horses in either experiment. Bacterial species isolated from treated horses were predominantly Pasteurella and/or Actinobacillus spp, however, members of the family Enterobacteriaceae and a Staphylococcus sp were isolated from treated horses. One treated horse in experiment A developed clinically apparent pulmonary disease. CONCLUSIONS: The prophylactic administration of penicillin before or during confinement did not reliably reduce bacterial numbers or prevent the accumulation of purulent lower respiratory tract secretions in horses confined with their heads elevated. Numbers of beta-haemolytic Streptococcus spp were reduced following treatment, suggesting that the repeated administration of procaine penicillin may have some merit as part of a strategy to prevent transport-associated respiratory disease. However, methods directed at minimising the duration of confinement with head elevation, augmentation of the clearance of accumulated secretions and prompt identification of animals in which airway inflammation has extended to the pulmonary parenchyma remain the best ways of minimising transport-associated respiratory disease.

Role of relative humidity in the survival of airborne Mycoplasma pneumoniae.

Aerosols of Mycoplasma pneumoniae were studied at several relative humidities at a controlled temperature of 27 C. Production of an experimentally reproducible aerosol required preatomization of the organism in its suspending fluid and was dependent on the type of fluid used in atomization as well as on the procedures used to produce an aerosol. The airborne particles studied were within the range of epidemiological significance, with most being 2 mum or less in diameter. Survival of the airborne mycoplasma in these particles was found to be best at very low and at very high humidities. The most lethal relative humidity levels were at 60 and 80%, at which levels fewer than 1% of the organisms survived over a 4-hr observation period. However, survival of the organism at most relative humidity levels was such that long-term infectivity could be expected from aerosols of M. pneumoniae. Because of the extreme sensitivity of M. pneumoniae at critical humidity levels, control of the airborne transmission of these organisms may be possible in selected spaces.

Fusobacterium equinum sp. nov., from the oral cavity of horses.

Two strains of gram-negative, anaerobic, non-sporulating rod that were isolated from the normal oral cavity and oral-associated disease from horses and which phenotypically resembled Fusobacterium necrophorum were characterized by sequencing of the 16S rRNA gene, phylogenetic analysis, DNA-DNA hybridization and phenotypic characterization. The results placed the novel strains as distinct members of the genus Fusobacterium. The novel species Fusobacterium equinum sp. nov. is proposed, with strain VPB 4027T (= NCTC 13176T = JCM 11174T) as the type strain.