Prognostic value of baseline absolute lymphocyte concentration and neutrophil/lymphocyte ratio in dogs with newly diagnosed multi-centric lymphoma.

Canine multi-centric B-cell lymphoma shares similarities with diffuse large B-cell (Non-Hodgkin's) lymphoma (NHL) in people. In people with NHL, lymphopenia at diagnosis and first relapse and neutrophil/lymphocyte ratio (N:L) > 3.5 are negative prognostic factors for survival. The objective of this study was to determine if lymphocyte concentration at diagnosis and first relapse and N:L were prognostic for survival in dogs with newly diagnosed multi-centric lymphoma. Medical records of 77 dogs with multi-centric lymphoma treated with a CHOP-based chemotherapy protocol were retrospectively evaluated. Absolute lymphocyte concentration and N:L ratio at presentation of dogs pre-treated with steroids was not significantly different from dogs who had not received steroids. On multivariate analysis, only immunophenotype remained significant for progression-free survival (PFS), whereas no variables remained significant for ST. A prospective study of these haematologic variables is warranted to assess their true significance.

Mycobacterium fortuitum lipoid pneumonia in a dog.

A 1-year old female spayed German Shepherd dog was evaluated for acute onset of dyspnea. Pyogranulomatous inflammation and green globoid structures were present on aspirates of the affected lung. Impression smears and histopathology confirmed pyogranulomatous pneumonia, with large amounts of lipid corresponding to the green structures noted cytologically, and identified poorly staining bacterial rods within lipid vacuoles. Special stains confirmed the presence of acid-fast bacterial rods, and polymerase chain reaction and DNA sequencing identified the organism as Mycobacterium fortuitum. M. fortuitum pneumonia is well described in humans and has previously been reported in 4 dogs and 1 cat. Lipid was a prominent cytologic and histologic feature, as is often described in humans and in the single feline case report. Additionally, this case highlights the variable cytologic appearance of lipid, as well as Mycobacterium spp, which are classically nonstaining with Wright-Giemsa.

PCR detection of Anaplasma platys in blood and tissue of dogs during acute phase of experimental infection.

Four dogs were experimentally infected with Anaplasma platys to determine changes in real-time TaqMan PCR detection in blood and tissue, microscopically detectable parasitemia, and platelet concentrations during the first 28 days of infection. Buffy-coat blood cells were PCR positive for A. platys DNA at 4 days after inoculation and remained positive in all dogs until day 14. Marked thrombocytopenia and low parasitemia occurred in dogs during that initial period. During 17 and 28 days post-inoculation, the PCR results on buffy-coat blood cells were intermittently negative in each dog with marked thrombocytopenia and no microscopic evidence of parasitemia. Bone marrow and splenic aspirates collected from the A. platys-infected dogs were tested by real-time TaqMan PCR. Two dogs were PCR positive in spleen and marrow at 28 days post-inoculation, when PCR results for buffy-coat blood cells were negative. Spleen and/or bone marrow samples should be considered as additional samples for PCR testing of dogs, particularly when blood samples are PCR negative during the acute phase of A. platys infection.

Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs.

BACKGROUND: Ineffective clearance of Ehrlichia canis after doxycycline administration has been reported despite the fact that the recommended treatment for canine ehrlichiosis is doxycycline. The effectiveness of doxycycline in clearing E canis infection from the blood and tissues of dogs requires additional evaluation. HYPOTHESIS: Doxycycline (5 mg/kg PO q12h), administered for 4 weeks, will eliminate E canis infection from the blood and tissues of experimentally infected dogs. ANIMALS: Fifteen Walker hound-mixed breed dogs were inoculated subcutaneously with E canis-infected canine histiocytic cells 4 months before doxycycline treatment. METHODS: Four dogs were treated with doxycycline (5 mg/kg PO q12h for 3 weeks), 5 dogs were treated with doxycycline at the same dosage for 4 weeks, and 5 control dogs were not treated. Dexamethasone (0.4 mg/kg i.v.) was given after treatment to precipitate recrudescence of any remaining E canis organisms. Platelet counts, anti-E canis immunofluorescent antibodies, and polymerase chain reaction (PCR) detection of E canis deoxyribonucleic acid (DNA) in blood and tissues were evaluated. RESULTS: E canis DNA was not detected in the blood and tissues of doxycycline-treated dogs after treatment. Platelet counts were within reference intervals, and E canis antibodies decreased. Spontaneous clearance of E canis infection occurred in 2 of 5 control dogs. Three control dogs had E canis DNA detected in blood and tissues, platelet counts remained low or within the reference interval, and E canis antibodies remained high. CONCLUSIONS AND CLINICAL IMPORTANCE: As administered in this study, doxycycline cleared E canis from the blood and tissues of experimentally infected dogs.

Failure of imidocarb dipropionate to clear experimentally induced Ehrlichia canis infection in dogs.

The recommended treatment for canine ehrlichiosis is tetracycline or its analog doxycycline, although recent reports have documented ineffective clearing of Erchlichia canis after doxycycline administration. Imidocarb dipropionate is used as an alternative treatment to tetracycline or is used in conjunction with doxycycline. The effectiveness of imidocarb dipropionate in clearing Ehrlichia species from the blood and tissues of dogs with E. canis infection has not been thoroughly evaluated. Fifteen dogs were experimentally infected with E. canis. Ten dogs were treated with imidocarb dipropionate (6.6 mg/kg, IM, 2 injections given 2 weeks apart). Five infected control dogs were not treated. Blood samples from all 15 dogs were E. canis DNA positive by PCR assay by 3 weeks after inoculation (PI), and E. canis antibodies were detected by IFA assay by 1 week PI. Blood platelet counts in all dogs were below the reference interval by 4 weeks PI. E. canis DNA was detected in bone marrow and splenic aspirates by PCR assay 4 weeks PI but not before infection. Bone marrow aspirates were E. canis DNA positive by PCR assay in 14/15 dogs, and splenic aspirates were E. canis DNA positive by PCR assay in 13/15 dogs. Blood samples from all treated and control dogs remained positive for E. canis DNA by PCR assay, and platelet counts remained below preinoculation values 13 weeks PI (6 weeks after 2nd treatment). As administered in this study, imidocarb dipropionate did not clear experimental E. canis infection in dogs.

Characterisation of the expression of a novel constitutive maize promoter in transgenic wheat and maize.

A novel constitutive promoter from the maize histone H2Bgene was recently identified. In this study, we characterised H2B promoter activity in both wheat and maize tissues using the gusA reporter gene and two synthetic versions of the pat (phosphinothricin acetyl transferase) selectable marker gene, namely mopat and popat. Analyses of transgenic plants showed that the H2B promoter is able to drive the expression of gusA to strong, constitutive levels in wheat and maize tissues. Using an H2B:mopat construct and phosphinothricin selection, we recovered transgenic wheat plants at efficiencies ranging from 0.3% to 7.4% (mean 1.6%), and the efficiency of selection ranged from 40% to 100% (mean 77.7%). In another application, H2B was combined with the maize Ubi-1 or the maize Adh-1 intron to drive the expression of mopat and popat. Transformation efficiencies with the Ubi-1 intron were between 1.4- to 16-fold greater than with the Adh-1 intron. However, the use of either of the introns was necessary for the recovery of transgenic plants. Mopat gave higher transformation efficiencies and induced higher levels of PAT protein in maize tissues than popat.

Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses.

We hypothesised that horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) react to an allergen or allergens in their summer environment that is either absent or present at lower levels in their winter environment; and that such allergens stimulate SPAOPD-affected horses to produce a different T helper lymphocyte cytokine profile from that of control horses. The primary objective of this study was to determine the cytokine mRNA profile of T helper lymphocytes obtained from summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses when 1) the horses were showing signs of disease (summer) and 2) they were in clinical remission (winter). A further objective was to determine the differences between cytokine mRNA T helper lymphocyte profiles of control and affected horses in the summer and winter seasons. Interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression levels were increased in bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cell (PBMC) samples of affected horses during disease expression. No significant amounts of IL-5 mRNA were detected in any of the samples. These results suggest that there is an allergic component to SPAOPD of horses and that appropriate manipulation of the immune system could offer hope for treatment and prevention of the disease in the future. Further research studies will be needed to determine the most appropriate treatments to use to alter the antigen-stimulated cytokine profile being expressed by SPAOPD-affected horses or to alter the effects that these cytokines produce.

Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment.

Ten current European wheat varieties were transformed at efficiencies ranging from 1-17% (mean 4% across varieties) following modifications in particle bombardment and tissue culture procedures. All plants surviving phosphinothricin selection were screened for uidA and bar gene activity, and for the presence of marker gene sequences by PCR analysis. A minimum of 35% plant 'escape' frequency was achieved with selection on 4 mg l(-1) gluphosinate ammonium after shoot initiation. Mean co-transformation frequency with various genes-of-interest was 66%. The estimated number of insertions of the uidA gene in 25 lines were; 1-2 in 32%, 3-5 in 52%, and 6-8 in 16% of lines. In T(1) progenies, marker genes segregated in a Mendelian fashion in 50% of 39 lines analysed, as determined by transgene activity assays. Based on PCR analysis, it appeared that in some lines the occurrence of distorted segregation was due to poor transmission of the transgenes.

Gradients and forward spreading of vertebrate Hox gene expression detected by using a Hox/lacZ transgene.

Elucidation of the kinetics with which vertebrate Hox expression patterns develop may help us to choose between various models already proposed to explain this process. The chick Hoxa-7/lacZ transgene, expressed in mouse embryos, changes over time in the distribution of its activity along the developing posterior to anterior axis. During an establishment (E) phase (lasting at least up to 10 days) expression is graded from highest levels posteriorly, to low levels anteriorly. Within the graded domain, the overall level of expression spreads forward with time along both neurectoderm and paraxial mesoderm. Spreading in expression is not due to movement of cells, but to increases in both the proportion of lacZ expressing cells and the intensity of expression per cell. By 10.8 days, embryos have reached a late (L) phase in which an anterior up-regulation in expression, together with a posterior down-regulation, cause the graded nature of the expression to be lost. E and L phases are also seen for Hox gene expression detected by in situ hybridization. The switch from E to L occurs at progressively later times as we move 3' to 5' along the Hox cluster. The results are in keeping with models in which Hox genes become differentially expressed according to a graded concentration of an inducer. Binding motifs for the caudal (cdx) proteins, already proposed as such inducers, are conserved in mouse and chick Hoxa-7 enhancer elements.

Transcutaneous blood gas monitoring in the rat.

Transcutaneous blood gas (TCBG) analysis is a noninvasive alternative method of estimation of blood gas tensions. The objective of the study reported here was to validate this method against standard blood gas (STBG) analysis in adult and juvenile Sprague-Dawley rats. We sought to establish the optimal TCBG probe site and temperature, to establish probe temperatures that would not cause thermal burns, to evaluate correlations between blood gas values (PaCO2 and PaO2) determined by use of TCBG and STBG, and to evaluate the sensitivity of the TCBG unit to changes in arterial blood gas partial pressures. Our results indicated that: in general, the xyphoid area was the optimal site for probe placement, with 44.5 degrees C being the optimal probe temperature for the highest correlation, but thermal burns may be a problem; probe temperatures of 42.5 degrees C (adults) and 42.0 degrees C (juveniles) do not cause thermal burns when left in place for three hours; probe temperatures of 44 degrees C (adults) and 42 degrees C (juveniles) resulted in moderate correlation between PaCO2 and PtcCO2; and the TCBG unit adequately responded to changes in arterial blood gas partial pressures. Neither PtcCO2 or PtcO2 reflect actual values of PaCO2 or PaO2, respectively. We concluded that TCBG analysis may be used as an indicator of change in PaCO2 with sufficient animal numbers under tightly controlled conditions, but not as an indicator of change in PaO2 in adult and juvenile rats.

Corticosteroid-responsive thrombocytopenia in two beef cows.

Corticosteroid-responsive thrombocytopenia was identified in 2 beef cows. Clinical findings in 1 cow included hematoma formation, petechiation of mucous membranes, anemia, and persistent thrombocytopenia. Cow 2 was in its fourth month of gestation and had epistaxis, nasal mucosal petechiation, anemia, and thrombocytopenia. Treatment included parenteral administration of corticosteroids at immunosuppressive dosages. Cow 1 had a history of chronic hematoma formation and responded to long-term treatment with dexamethasone, but it relapsed 2 months after treatment was discontinued. Cow 2 had acute onset of clinical signs, responded to short-term treatment with prednisone, delivered a full-term, healthy calf, and remained clinically normal for at least 1 year after treatment was completed. Reported causes of thrombocytopenia in ruminants were ruled out or seemed unlikely; a definitive cause for thrombocytopenia in the 2 cows could not be established.

Evolutionary shifts of vertebrate structures and Hox expression up and down the axial series of segments: a consideration of possible mechanisms.

The term 'transposition' describes how, during vertebrate evolution, anatomical structures have shifted up or down the axial series of segments. For example, the neck/thorax junction and the position of the forelimb in the chicken have shifted posteriorly, relative to mouse, by a distance of seven somites or vertebrae. By examining the expression boundaries of some chick Hox genes not previously described, we provide new evidence that axial shifts in anatomical structures correspond with shifts in Hox expression domains. These shifts occur both in mesodermal components (somites, vertebrae, and lateral plate mesoderm) and neural components (spinal ganglia). We discuss morphogen gradient, timing, spreading, and growth models for the setting of Hoxexpression boundaries, and consider how evolutionary shifts in boundary positions might have been effected in terms of these models.

Correlation of clinical score, intrapleural pressure, cytologic findings of bronchoalveolar fluid, and histopathologic lesions of pulmonary tissue in horses with summer pasture-associated obstructive pulmonary disease.

OBJECTIVE: To correlate clinical score, intrapleural pressure, cytologic findings of bronchoalveolar lavage fluid (BALF), and histologic lesions of pulmonary tissue in horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD). ANIMALS: 8 adult horses affected with SPAOPD and 6 adult horses without evidence of respiratory tract disease. PROCEDURE: Clinical score, change in intrapleural pressure (deltaPpl) during tidal breathing, results of cytologic examination and bacteriologic culture of BALF, and results of histologic examination of pulmonary parenchyma were evaluated. RESULTS: Clinical scores for SPAOPD-affected horses (median, 5.75; range, 4.0 to 7.5) were significantly greater, compared with clinically normal horses (median, 2.0; range, 2.0 to 3.0). Cytologic examination of BALF from SPAOPD-affected horses revealed predominantly nondegenerate neutrophils. Histologic lesions were identified throughout pulmonary tissue and included severe accumulation of mucus and neutrophils within the small airways, metaplasia of bronchiolar goblet cells, and mild peribronchial infiltrate. Histologic examination of specimens collected via percutaneous biopsy was predictive of disease and corresponded to findings at postmortem examination. Clinical score and deltaPpl were highly correlated with mucus accumulation in the airways of affected horses. Peribronchial inflammatory infiltrate correlated with percentage of neutrophils in BALF of affected horses. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical scoring and deltaPpl provided valid estimates of disease severity. Findings from cytologic examination of BALF of SPAOPD-affected horses varied, although, in most instances, it was diagnostically useful. Severe mucus accumulation in the airways was the most remarkable histopathologic finding in SPAOPD-affected horses. Examination of biopsy specimens collected from pulmonary parenchyma was consistently useful in diagnosing SPAOPD.

Detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia.

Horse mares carrying mule foals were immunized during the last trimester of pregnancy with whole acid-citrate-dextrose-anticoagulated donkey blood to experimentally induce neonatal alloimmune thrombocytopenia. Thrombocytopenia occurred in the neonatal mule foals born to immunized horse mares within 24 hours after ingestion of their dams' colostrum. Mule foals born to mares not immunized with donkey blood did not develop thrombocytopenia. These findings suggest that antibodies may have been directed against a donkey platelet antigen present in the mule foals but not present in their dams. The objectives of this study were to determine whether anti-platelet antibody could be detected in mule foals with experimentally induced neonatal alloimmune thrombocytopenia, to identify any platelet proteins recognized by serum antibody in these foals, and to determine if platelet function was altered by sera from these mule foals. An indirect enzyme-linked immunosorbent assay demonstrated significantly higher absorption at 1:200 of platelet-bindable immunoglobulin G in serum from thrombocytopenic mule foals, compared with nonthrombocytopenic mule foals. Sera from thrombocytopenic and nonthrombocytopenic mule foals produced similar binding patterns in western immunoblots with donkey platelet proteins separated on sodium dodecyl sulfate polyacrylamide gels. Maximal platelet aggregation and relative slope of aggregation in response to collagen were significantly inhibited after incubation with sera from thrombocytopenic mule foals. These results suggest that mule foals with induced alloimmune thrombocytopenia have serum antibodies that bind to platelets and may compete with collagen binding sites to impair platelet aggregation.

Evidence that Hoxa expression domains are evolutionarily transposed in spinal ganglia, and are established by forward spreading in paraxial mesoderm.

Transposition of anatomical structures along the anteroposterior axis has been a commonly used mechanism for changing body proportions during the course of evolutionary time. Earlier work (Gaunt, S.J., 1994. Conservation in the Hox code during morphological evolution. Int. J. Dev. Biol. 38, 549-552; Burke, A.C., Nelson, C.E., Morgan, B.A., Tabin, C., 1995. Hox genes and the evolution of vertebrate axial morphology. Development 121, 333-346) showed how transposition in mesodermal derivatives (vertebrae) could be attributed to transposition in the expression of Hox genes along the axial series of somites. We now show how transposition in the segmental arrangement of the spinal nerves can also be correlated with shifts in the expression domains of Hox genes. Specifically, we show how the expression domains of Hoxa-7, a-9 and a-10 in spinal ganglia correspond similarly in both mouse and chick with the positions of the brachial and lumbosacral plexuses, and that this is true even though the brachial plexus of chick is shifted posteriorly, relative to mouse, by seven segmental units. In spite of these marked species differences in the boundaries of Hoxa-7 expression, cis regulatory elements located up to 5 kb upstream of the chick Hoxa-7 gene showed much functional and structural conservation with those described in the mouse (Puschel, A.W., Balling, R., Gruss, P., 1991. Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression. Development 112, 279-287; Knittel, T., Kessel, M., Kim, M.H., Gruss, P., 1995. A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development. Development 121, 1077-1088). We also show that chick Hoxa-7 and a-10 expression domains spread forward into regions of somites that are initially negative for the expression of these genes. We discuss this as evidence that Hox expression in paraxial mesoderm spreads forward, as earlier found for neurectoderm and lateral plate mesoderm, in a process that occurs independently of cell movement.

Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues.

The objective of this study was to identify the major parameters controlling DNA delivery by particle bombardment to wheat (Triticum aestivum L.) scutellum and inflorescence tissue. The main factors studied were the DNA/gold precipitation process, bombardment parameters and tissue culture variables. Efficiency of DNA (uidA gene) delivery was assessed by scoring transient GUS expression in bombarded tissues. Of the parameters analysed, amount of plasmid DNA, spermidine concentration, presence of Ca++ ions, calcium chloride concentration, amount of gold particles, gold particle size, acceleration pressure, chamber vacuum pressure, bombardment distance, osmotic conditioning of tissues and type of auxin had a clear influence on transient gene expression. A bombardment procedure suitable for elite wheat varieties was developed which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.