Flow cytometry in veterinary oncology.

Flow cytometry is a highly sensitive and specific method for simultaneous analysis of multiple parameters of individual cells in a suspension. It has a range of applications in veterinary medicine, and it is increasingly used in veterinary oncology as more species-specific antibodies are generated and cross-reactivity of antibodies is characterized. Two major applications in veterinary oncology are (1) immunophenotyping with a panel of fluorescently labeled antibodies to assess expression of cell markers and (2) determination of the DNA content of cells with fluorescent dyes that bind nucleic acids. The diagnostic and prognostic value of classifying round cell tumors of animals-especially, lymphocyte proliferations-remains to be fully determined, but studies to date have indicated benefit to patient management. Similarly, determining the proliferating fraction of tumors through DNA analysis remains to be standardized and validated in veterinary oncology but shows promise as an adjunct to morphologic tumor classification. This article reviews technical aspects of flow cytometry, availability of antibodies suitable for studies in domestic animals, and applications in veterinary oncology with emphasis on characterization of round cell tumors.

Alloimmunity does not protect from challenge with the feline immunodeficiency virus.

Immune responses against polymorphic host molecules incorporated into lentiviral envelopes during cell budding have induced protection against primate immunodeficiency virus infection. Dendritic cells (DCs) express high levels of MHC molecules and are infectable by lentiviruses. Therefore, in this pilot study we addressed the hypothesis that immunization of cats with allogeneic DC would induce immune responses that protect against challenge with the feline immunodeficiency virus. Two groups of 3 cats each received 3 subcutaneous injections of allogeneic or autologous DC, and were then challenged with viruses propagated in the immunizing DC. Infection status and lymphocyte parameters of cats were assessed during 6 weeks after challenge. MHC II antigens were incorporated into viral particles as identified by Western blot; and antibodies reactive with MHC class II antigens were detected in the serum of cats immunized with allogeneic but not autologous DC. After challenge, all cats had proviral DNA in blood leukocytes from 2 weeks post-challenge onward and seroconverted. Cats immunized with allogeneic DC maintained higher total and CD21(+) lymphocyte concentrations, and higher CD4(+)/CD8(+) lymphocyte ratios; however, these differences were not significantly different from cats that received autologous DC immunizations. Plasma viral load was not significantly different between groups of cats (p=0.204). These results suggest that immunization of cats with allogeneic DC does not induce protective immunity against FIV infection.

CD134 and CXCR4 expression corresponds to feline immunodeficiency virus infection of lymphocytes, macrophages and dendritic cells.

The lymphotropic lentiviruses feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) enter cells by sequential interaction with primary receptors CD134 or CD4, respectively, and subsequently with chemokine receptors. The host-cell range for FIV is broader than that for HIV, but whether this is a function of receptor expression is unknown. Lack of reagents specific to feline molecules has limited detection and analysis of receptors and their interaction with viral components. Here, the expression of CD134 and CXCR4 on feline T and B lymphocytes, dendritic cells (DCs) and macrophages was examined and the kinetics of FIV replication were assessed. Quantification of CD134 mRNA by real-time PCR indicated expression in all leukocytes, with significantly more transcripts in CD4(+) lymphocytes than in other leukocytes. Antibodies against human CD134 bound inconsistently to feline leukocytes. CXCR4 was detected with antibody clone 12G5 on the surface of monocyte-derived cells only, but gene transcripts were present in all cells, with the highest copy number in lymphocytes. CXCR4 expression decreased and CD134 expression increased with cell activation in lymphocytes. A subtype B biological isolate of FIV infected DCs, macrophages and lymphocytes, with the highest replication in CD4(+) lymphocytes, whilst cloned FIV P14 infected all cells, but replicated less efficiently. Although viral replication was lower in DCs and macrophages than in lymphocytes, DCs expressed specific receptors and were infected productively with FIV, as indicated by viral ultrastructure and DNA detection. These results may implicate altered function of DCs in the induction of specific immunity against FIV.

Invasive epithelial mesothelioma in a dog.

This report describes the gross, microscopic, and immunohistochemical features of an invasive epithelial mesothelioma in an 11-year-old neutered male Golden Retriever. The tumor involved the pericardium, pleura, mediastinum, and peritoneum and invaded into submesothelial tissues. Neoplastic cells in the thoracic fluid showed prominent features of malignancy in a background of mixed inflammatory cells and scattered erythrocytes. Histologically, the tumor consisted of nests of epithelioid cells with frequent mitotic figures and multinucleation that infiltrated submesothelial tissues. Neoplastic cells strongly coexpressed vimentin and cytokeratin intermediate filaments, which assisted in the differentiation from other epithelial tumors of nonmesothelial origin.

The variability of serological and molecular diagnosis of feline immunodeficiency virus infection.

Diagnosis of feline immunodeficiency virus (FIV) infection by polymerase chain reaction (PCR) has recently become available, but little is known about the performance of this assay. The purpose of this study was to determine the sensitivity and specificity of PCR diagnosis of FIV infection. Replicate aliquots of blood samples from cats identified as FIV positive or negative by 2 previous enzyme-linked immunosorbent assay (ELISA) results, and from clinically healthy dogs, were submitted to different laboratories for FIV serologic diagnosis and PCR. The PCR products obtained in 1 laboratory were sequenced to determine the FIV subtype. The PCR assays correctly identified 100%, 80%, and 50% of the FIV-positive samples, and 100%, 90%, and 70% of FIV-negative samples. Each dog sample was reported as FIV PCR positive at least once, and FIV subtypes A, B, and C were identified. It was concluded that PCR tests currently available for FIV infection are unreliable, with highly variable sensitivity and specificity.

Feline immunodeficiency virus subtypes A, B and C and intersubtype recombinants in Ontario, Canada.

Knowledge of the geographical distribution of feline immunodeficiency virus (FIV) subtypes is important for understanding different disease courses and for vaccine design. Intersubtype recombination may develop in areas where more than one subtype is prevalent and has the potential to create new transmittable variants with novel pathogenic properties. In this study, 40 FIV-positive DNA samples were classified by sequence analysis of the LTR-gag region. Phylogenetic analysis indicated that 32 Canadian FIV isolates clustered with previously identified subtypes A, B and C and that subtype A was most frequent in Ontario. Four strains with inconsistent clade assignment were further analysed by sequencing of the env-LTR regions. Comparisons of phylogenetic trees constructed from the two different regions of the genome and analysis of similarities to reference sequences yielded classification of three samples as A/B and one as A/C intersubtype recombinants. Although the A/B recombinant samples were obtained from unrelated cats in geographically disparate regions, a common breakpoint was consistently identified within gag. In addition, there was no evidence of co-infection with parental strains of subtypes A and B as indicated by PCR-based limiting dilution assays, although these assays allowed for the identification of two different recombinant viruses co-existing in one sample. Both sequences contained the same breakpoint. These findings suggested that a new circulating recombinant FIV may be enzootic in Ontario.

Immunophenotype and functional properties of feline dendritic cells derived from blood and bone marrow.

Dendritic cells (DCs) are a heterogeneous population of cells of fundamental importance in initiating innate as well as specific immune responses. The identity and function of DCs in the cat are unknown, although they are likely pivotal in the response to infection. In this study, feline DCs were derived by 3-10-day culture of adherent blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) in the presence of IL 4 and GM-CSF. BMMC consistently yielded a greater number of DCs than PBMC, and there were fewer macrophages than DC from both compartments. DCs expressed a distinct constellation of surface molecules, which included CD1a, CD1b, and CD1c, CD11b, CD14, and 2-3-fold higher levels of MHC class I and II molecules than co-cultured macrophages or fresh blood monocytes. DCs displayed typical cytoplasmic processes, limited non-specific esterase activity, and acquired antigen by phagocytosis, pinocytosis, and binding to specific receptors. Cytokine-exposed cells induced proliferation of allogeneic lymphocytes. Thus, the cells derived by these culture conditions had markers and functions analogous to immature myeloid DCs. Availability of feline DCs will enable investigation of their role in infectious disease and their potential therapeutic application.

Equine rotaviruses with G14 serotype specificity circulate among venezuelan horses.

Two group A rotavirus strains isolated from diarrheic foals in Venezuela were classified as belonging to G14 serotype by cross-neutralization tests and on the basis of the homology of the sequenced VP7 gene. This report confirms that rotavirus strains of G14 serotype specificity circulate among equine populations.