Proposed criteria for classification of acute myeloid leukemia in dogs and cats

Blood and bone marrow smears from 49 dogs and cats, believed to have myeloproliferative disorders (MPD), were examined by a panel of 10 clinical pathologists to develop proposals for classification of acute myeloid leukemia (AML) in these species. French-American-British (FAB) group and National Cancer Institute (NCI) workshop definitions and criteria developed for classification of AML in humans were adapted. Major modifications entailed revision of definitions of blast cells as applied to the dog and cat, broadening the scope of leukemia classification, and making provisions for differentiating erythremic myelosis and undifferentiated MPD. A consensus cytomorphologic diagnosis was reached in 39 (79.6%) cases comprising 26 of AML, 10 of myelodysplastic syndrome (MDS), and 3 of acute lymphoblastic leukemia (ALL). Diagnostic concordance for these diseases varied from 60 to 81% (mean 73.3 +/- 7.1%) and interobserver agreement ranged from 51.3 to 84.6% (mean 73.1 +/- 9.3%). Various subtypes of AML identified included Ml, M2, M4, M5a, M5b, and M6. Acute undifferentiated leukemia (AUL) was recognized as a specific entity. M3 was not encountered, but this subclass was retained as a diagnostic possibility. The designations M6Er and MDS-Er were introduced where the suffix "Er" indicated preponderance of erythroid component. Chief hematologic abnormalities included circulating blast cells in 98% of the cases, with 36.7% cases having >30% blast cells, and thrombocytopenia and anemia in approximately 86 to 88% of the cases. Bone marrow examination revealed panmyeloid dysplastic changes, particularly variable numbers of megaloblastoid rubriblasts and rubricytes in all AML subtypes and increased numbers of eosinophils in MDS. Cytochemical patterns of neutrophilic markers were evident in most cases of Ml and M2, while monocytic markers were primarily seen in M5a and M5b cases. It is proposed that well-prepared, Romanowsky-stained blood and bone marrow smears should be examined to determine blast cell types and percentages for cytomorphologic diagnosis of AML. Carefully selected areas of stained films presenting adequate cellular details should be used to count a minimum of 200 cells. In cases with borderline diagnosis, at least 500 cells should be counted. The identity of blast cells should be ascertained using appropriate cytochemical markers of neutrophilic, monocytic, and megakaryocytic differentiation. A blast cell count of > 30% in blood and/or bone marrow indicates AML or AUL, while a count of < 30% blasts in bone marrow suggests MDS, chronic myeloid leukemias, or even a leukemoid reaction. Myeloblasts, monoblasts, and megakaryoblasts comprise the blast cell count. The FAB approach with additional criteria should be used to distinguish AUL and various subtypes of AML (Ml to M7 and M6Er) and to differentiate MDS, MDS-ER, chronic myeloid leukemias, and leukemoid reaction. Bone marrow core biopsy and electron microscopy may be required to confirm the specific diagnosis. Immunophenotyping with lineage specific antibodies is in its infancy in veterinary medicine. Development of this technique is encouraged to establish an undisputed identity of blast cells. Validity of the proposed criteria needs to be substantiated in large prospective and retrospective studies. Similarly, clinical relevance of cytomorphologic, cytochemical, and immunophenotypic characterizations of AML in dogs and cats remains to be determined.     

An improved ferritin assay for canine sera

An improved serum ferritin assay for canine serum has been developed. It uses two monoclonal antibodies in a sandwich arrangement. Serum ferritin can be determined on undiluted canine sera with this assay. The recovery of ferritin added to canine serum ranged from 98 to 106%, the within-assay coefficient of variability was 3.3 to 4.5%, and the assay-to-assay variability was 9.8 to 10.2%. Serum ferritin from 61 apparently healthy dogs had a geometric mean of 252 ng/ml, with a range of 80 ng/ml to 800 ng/ml.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Characteristics for Practical Use of Attenuated Isolate UA-Fukushima of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis>

L₁₁A-Fukushima (L₁₁A-F) derived from attenuated isolate LuA of Tomato mosaic virus (ToMV) has the highest ability to cross protect against virulent ToMV among LuA and its derivatives and is stably inherited. Growth, yield, fruit quality and symptom attenuation of inoculated tomato plants did not differ significantly between L₁₁A-F and L₁₁A. The infectivity of progeny viruses in tomato infected with LuA-F was less than 4% of that with virulent ToMV. From these results, L₁₁A-F appears to possess the properties necessary for practical use. To manage L₁₁A-F strictly, a PCR-based assay to detect trace contamination of virulent ToMV in L₁₁A-F preparations was established. Received 10 June 2002/ Accepted in revised form 30 October 2002     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

Importance of Cell Wall Degrading Enzymes Produced by Fusarium graminearum during Infection of Wheat Heads

Cytological studies were carried out to elucidate the importance of cell wall degrading enzymes (CWDE) during infection of wheat spikes by Fusarium graminearum. Scanning electron micrographs revealed that at 6–24 hours after inoculation (hai) of single spikelets with macroconidia of F. graminearum, the fungus germinated by forming several germ tubes and developed a dense hyphal network in the cavity of the spikelet. At 24–36hai, the fungus formed infection hyphae which invaded the ovary and inner surface of the lemma and palea. Transmission electron microscopical studies revealed that the fungus extended inter- and intracellularly in the ovary, lemma and rachis and caused considerable damage and alterations to the host cell walls. In different tissues of healthy and F. graminearum-infected wheat spikes the cell wall components cellulose, xylan and pectin were localized by means of enzyme-gold and immuno-gold labelling techniques. Localization of cellulose, xylan and pectin showed that host cell walls which were in direct contact with the pathogen surface had reduced gold labelling compared to considerable higher labelling densities of walls distant from the pathogen–host interface or in non-colonized tissues. The reduced gold labelling densities in the infected host cell walls indicate that these polysaccharide degrading enzymes might be important pathogenicity factors of F. graminearum during infection of wheat spikes. The results revealed that, infection and colonization of wheat spikes by F. graminearum and reactions of infected host tissue were similar to those reported for F. culmorum.     

Indole-3-acetic acid biosynthesis in <Emphasis Type="Italic">Aciculosporium take</Emphasis>, a causal agent of witches' broom of bamboo

 Aciculosporium take (Ascomycota; Clavicipitaceae) is a causal agent of witches' broom of bamboo plants. The symptoms of this disease are believed to be induced by plant hormones, particularly auxins. Indole-3-acetic acid (IAA) was identified in cultures of this fungus in an l-tryptophan-supplemented liquid medium. IAA production was confirmed on 30 isolates of A. take from various hosts and locations at levels up to 1 mg/l. The biosynthetic pathway of IAA in A. take culture was examined by analyzing intermediate products and by feeding experiments. The results showed that the indole-3-pyruvic acid pathway (l-tryptophan → indole-3-pyruvic acid → indole acetaldehyde → IAA) was the dominant pathway in A. take. Received: June 3, 2002 / Accepted: July 25, 2002