Detection of feline immunodeficiency virus infection in bone marrow of cats.

Natural or experimental feline immunodeficiency virus (FIV) infection in cats is often associated with hematologic abnormalities which are similar to those observed in human immunodeficiency virus (HIV) infected patients. To determine if cells in bone marrow are infected with FIV and whether severity of hematopoietic disorder is correlated with the level of viral infection, bone marrow tissues from ten experimentally and two naturally FIV infected cats were examined by in situ hybridization for presence of FIV RNA. Seven of the 12 FIV infected cats were also naturally or experimentally coinfected with feline leukemia virus (FeLV). FIV RNA was detected mainly in megakaryocytes and unidentified mononuclear cells in the bone marrow of cats that were sick and had marrow hypercellularity and immaturity. These included all cats in the acute phase of FIV infection and two of seven long term FIV infected cats. One long term FIV infected cat with lymphosarcoma was also positive for FIV RNA in bone marrow cells. The other four long term FIV infected cats were relatively healthy, with normal bone marrow morphology, and were negative for FIV infected cells. Bone marrow from three non-infected and two cats infected with FeLV alone were also negative for FIV RNA by in situ hybridization. We concluded that megakaryocytes and mononuclear cells were targets of the viral infection and that the presence of FIV RNA in cells of the bone marrow correlated with marrow hypercellularity and immaturity, and severity of illness.     

Characterization of fibroblast colony-forming units in bone marrow from healthy cats

The adherent cell layer of bone marrow from healthy cats was characterized in vitro, and the mean fibroblast colony-forming unit (CFU-F) was determined. The majority (82%) of the cells in the adherent cell layer were spindle-shaped fibroblastic cells. These cells were weakly positive for acid phosphatase activity and negative for alpha-naphthyl butyrate esterase and alkaline phosphatase activities. They did not phagocytose latex beads. The remaining cells (18%) in the adherent cell layer resembled macrophages. They were strongly positive for acid phosphatase and alpha-naphthyl butyrate esterase activities, and they phagocytosed latex beads. The mean CFU-F per 10(6) mononuclear cells in bone marrow from healthy kittens and adult cats was 62 and 65, respectively. The CFU-F assay was linear over a range of 0.25 to 1.25 x 10(6) bone marrow mononuclear cells cultured. Variation in the feline CFU-F assay was similar to that reported for the human CFU-F assay. Bone marrow collections repeated at 1-month intervals (from the same bone) did not affect CFU-F concentration. A difference was not observed between CFU-F cultured from the feline humerus or femur. Bone marrow adherent cells in cats resembled those described for other species. Results of the feline CFU-F assay were consistent and repeatable and were similar to those reported for other species.     

Suppression of feline bone marrow fibroblast colony-forming units by feline leukemia virus

Bone marrow fibroblast colony-forming units (CFU-F) were evaluated in cats experimentally infected with feline leukemia virus (FeLV). Cats that developed persistent viral infection and anemia (progressor cats) had a progressive decrease in the number of CFU-F at 2, 4, 6, 8, and 10 weeks after inoculation with FeLV. This suppression of CFU-F number in progressor cats ranged from 16 to 44% of the preinoculation CFU-F value. Cats that did not develop persistent viral infection or anemia (regressor cats) had decreased numbers of CFU-F (24% of the preinoculation CFU-F value) at 2 weeks after inoculation, but normal CFU-F numbers at 4, 6, 8, and 10 weeks after inoculation. In vitro incubation of bone marrow mononuclear cells from healthy cats with the 15,000-dalton envelope protein of FeLV resulted in decreased number of CFU-F (21% of that of untreated cultures). The number of CFU-F from bone marrow mononuclear cells incubated with the 27,000-dalton core protein of FeLV was similar to that from untreated cultures.     

A hematological study on thirteen cats with myelodysplastic syndrome

Hematological abnormalities were investigated in 13 cats with myelodysplastic syndrome (MDS). Examination of the peripheral blood samples from the 13 cats revealed anemia in 11 cats, leukopenia in 9 cats, and thrombocytopenia in 9 cats. Four cats had pancytopenia (30.8%) and 9 cats had bicytopenia (69.2%). Dysplastic changes of erythrocytes, neutrophils, and platelets in the peripheral blood were found in 5, 10 and 8 cats, respectively. Bone marrow examination of the 13 cats revealed that ratios of blast cells to all nucleated cells (ANC) ranged from 0 to 20%. Ratios of erythroid progenitor cells to ANC were more than 50% in 3 cats and less than 50% in 10 cats. Eosinophils accounted for more than 5% of non-erythroid cells in 10 cats. Dysplastic changes in the granurocytic, erythrocytic, and megakaryocytic cells in the bone marrow were found in 11, 7 and 5 cats, respectively. Dysplastic changes in these cats included giant neutrophils, ring-nucleated neutrophils, binuclear myelocytes, hypersegmented and hyposegmented neutrophils, megaloblastoid erythroblasts, multinucleated erythroblasts, micromegakaryocytes, and segmented multinucleated megakaryocytes. Virological examination indicated the presence of feline leukemia virus antigen in the peripheral blood from all of the 13 cats with MDS. The peripheral blood cytopenias and dysplastic changes in each blood cell lineage in the bone marrow were shown to be important for the diagnosis of MDS in cats.     

Feline leukemia virus-associated enteritis--a condition with features of feline panleukopenia

Infection with feline leukemia virus (FeLV) was demonstrated immunohistologically in 218 necropsied cats suffering from enteritis. The animals were divided into three groups according to histopathological criteria. The first group exhibited the signs of feline panleukopenia in intestine, lymphoid tissues, and bone marrow. Only 1.6% of these animals were FeLV-infected. The animals of the second group had histopathological alterations as seen in cats suffering from feline panleukopenia, but these were found only in the intestine and not in lymphoid tissues or bone marrow. Of these 71.9% were infected with FeLV. The third group consisted of all other cats suffering from enteritis of which 6.3% were FeLV-positive. The association between FeLV infection and the lesions seen in the animals of group 1 (feline panleukopenia) and group 3 (other types of enteritis) is statistically not significant whereas the alterations exhibited by the cats of group 2 are significantly FeLV-associated. Cats with FeLV-associated enteritis (group 2) are of a mean age of about 2.5 years and are significantly older than animals with feline panleukopenia which are of a mean age of about half a year. Thus a FeLV-associated enteritis exists as a histopathologically recognizable condition which sometimes might be mistaken for feline panleukopenia in routine post-mortem investigations.     

In vitro selective suppression of feline myeloid colony formation is attributable to molecularly cloned strain of feline leukemia virus with unique long terminal repeat

Molecularly cloned feline leukemia virus (FeLV)-clone 33 (C-33), derived from a cat with acute myelocytic leukemia (AML), was examined to assess its relation to the pathogenesis of AML and myelodysplastic syndrome (MDS). To evaluate in vitro pathogenicity of FeLV C-33, bone marrow colony-forming assay was performed on marrow cells infected with FeLV C-33 or an FeLV subgroup A strain (61E, a molecularly cloned strain with minimal pathogenicity). The myeloid colony-forming activity of feline bone marrow mononuclear cells infected with FeLV C-33 was significantly lower than that of cells infected with 61E. This suggests that FeLV C-33 has myeloid lineage-specific pathogenicity for cats, and that FeLV C-33 infection is useful as an experimental model for investigating pathogenesis of MDS and AML.     

Identification of parvovirus in the bone marrow of eight cats

Objective To determine if canine parvovirus (CPV) or feline panleucopenia virus (FPV) genomic sequences are present in adult feline bone marrow samples. Design Bone marrow samples were obtained from 32 semi‐feral cats that were euthanased at an animal shelter. DNA was extracted and subjected to conventional polymerase chain reaction (PCR) designed to determine if CPV or FPV DNA was present. Positive PCR products were purified, cloned and sequenced to differentiate between CPV and FPV. Results Eight of the bone marrow samples contained parvoviral DNA (7 CPV, 1 FPV). Conclusion CPV and FPV DNA can be found in the bone marrow of healthy adult cats.     

Incidence of persistent viraemia and latent feline leukaemia virus infection in cats with lymphoma

In the past, feline leukaemia virus (FeLV) infection, and also latent FeLV infection, were commonly associated with lymphoma and leukaemia. In this study, the prevalence of FeLV provirus in tumour tissue and bone marrow in FeLV antigen-negative cats with these tumours was assessed. Seventy-seven diseased cats were surveyed (61 antigen-negative, 16 antigen-positive). Blood, bone marrow, and tumour samples were investigated by two polymerase chain reaction (PCR) assays detecting deoxyribonucleic acid (DNA) sequences of the long terminal repeats (LTR) and the envelope (env) region of the FeLV genome. Immunohistochemistry (IHC) was performed in bone marrow and tumour tissue. None of the antigen-negative cats with lymphoma was detectably infected with latent FeLV. The prevalence of FeLV viraemia in cats with lymphoma was 20.8%. This suggests that causes other than FeLV play a role in tumorigenesis, and that latent FeLV infection is unlikely to be responsible for most feline lymphomas and leukaemias.     

Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus

Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula.     

Altered surface antigen expression on peripheral blood mononuclear cells in cats infected with feline immunodeficiency virus.

Expression of CD4, CD8, IL-2 receptor alpha chain (IL-2R alpha), and MHC class II (MHC-II) on peripheral blood mononuclear cells were examined in cats infected with feline immunodeficiency virus (FIV). CD4/CD8 T cell ratio in FIV-infected cats was slightly decreased, as compared with that in specific-pathogen-free (SPF) cats. However, there was no statistical differences between them. The number of circulating IL-2R alpha+ cells in FIV-infected cats was higher than that in healthy cats, whereas induction of IL-2R alpha expression by concanavalin A (Con A) stimulation was depressed in FIV-infected cats. By using two-color cytofluorometry, Con A-induced enhancement of IL-2R alpha expression was found to be reduced in both CD4+ and CD8+ populations in PBMC from FIV-infected cats. The circulating MHC-II+ cells were also increased in FIV-infected cats. Furthermore, the induction of IL-2R alpha expression on PBMC after Con A-stimulation significantly depressed by FIV inoculation in vitro. These results suggest that FIV activates PBMC in vivo via direct and/or indirect mechanisms, leading to the unresponsive state of T cells to further stimuli in vitro.     

Effects of treatment with cobra venom factor on experimentally induced feline leukemia

Five- to six-month-old specific-pathogen-free cats were exposed to cobra venom factor (CVF) alone (4 cats), Rickard feline leukemia virus (FeLV; 9 cats), or CVF and FeLV (6 cats). Host-virus relationships were evaluated by monitoring the development of viremia, production of antibody against feline oncornavirus-associated cell membrane antigen, and amount of circulating immune complexes (CIC). Exposure to CVF induced complement depletion, which lasted 8 to 15 days. However, complement depletion did not promote the development of persistent viremia nor alter the production of antibody to feline oncornavirus-associated cell membrane antigen or CIC. Results indicated that the complement system did not protect cats during their initial exposure to FeLV and that an intact complement system was not necessary for the development of antibody against feline oncornavirus-associated antigen or for the formation of CIC.     

Evaluation of P-glycoprotein expression in feline lymphoma and correlation with clinical outcome

P-glycoprotein (Pgp) is a transmembrane protein pump involved in drug resistance in canine and human lymphoma. There are no published clinical studies evaluating Pgp expression in feline lymphoma. The purpose of this study is to evaluate the level of Pgp expression in feline lymphoma and correlate it with clinical outcome. Two human Pgp monoclonal antibodies, C219 and C494, were used to detect Pgp expression in tissue samples from 63 cats with lymphoma. Demographic results appear comparable to recently published feline lymphoma studies. The Kaplan–Meier median remission and survival times were 164 and 571 days, respectively. Fourteen cats had positive expression of Pgp using MAb C219, and 40 were positive with C494. Variables statistically associated with survival included bone marrow involvement, stage, substage, and use of radiation therapy as a part of treatment. Pgp expression as assessed by MAb C219 and C494 is not predictive of remission or survival time in cats with lymphoma.     

Pathogenesis of feline gastric chlamydial infection

Studies were conducted to determine whether the gastric chlamydiae that have been observed recently in cats are of pathologic significance. Chlamydiae were isolated in mouse L cell cultures from the homogenized pooled gastric mucosa of 3 cats that had been identified, by histopathologic examination, to have gastric chlamydiosis. Ten specific-pathogen-free kittens were exposed by aerosol and oral inoculation to the harvested feline gastric chlamydiae cell-culture media. In general, the clinical signs and lesions were conjunctivitis, rhinitis, and mild gastritis. The clinical signs and lesions were most severe in 2 chlamydia-infected kittens that had received methylprednisolone acetate (50 mg/kg of body weight). Chlamydiae were demonstrated in epithelial cells of conjunctival and nasal smears in 10 of 10 infected kittens from postexposure days 7 through 35. In addition, chlamydiae were isolated in L cell cultures from a variety of antemortem and postmortem specimens from infected kittens. The present study provided evidence that feline gastric chlamydiae, under appropriate conditions, were capable of inducing, in cats, clinical signs and lesions similar to those induced by the feline pneumonitis agent.     

Retroviral-associated eosinophilic leukemia in the cat

Fourteen specific-pathogen-free cats were inoculated with a putative env gene recombinant feline retrovirus, PR8. An isolate of the Rickard strain of feline leukemia virus (FeLV-R), PR8, has the properties of both an exogenous (FeLV-R) and an endogenous (xenotropic) feline retrovirus (RD-114). Twelve of the PR8-inoculated cats developed viremia; 2 of the 12 cats developed eosinophilia, with 1 being diagnosed with eosinophilic leukemia and the other with extreme eosinophilic hyperplasia. Eosinophilic leukemia is rare in cats and has not previously been associated with retroviral infection. Changes in the viral envelope properties may have altered the pathogenicity of the exogenous virus to cause this rare form of leukemia.     

Aplastic anemia in cats - clinicopathological features and associated disease conditions 1996-2004

A retrospective study of 128 feline bone marrow reports identified 13 cases of aplastic anemia. Clinical diagnoses included chronic renal failure (n=5), feline leukemia virus infection (n=2), hyperthyroidism treated with methimazole (n=1) and idiopathic aplastic anemia (n=5). In some cats, starvation may play a role in the development of marrow aplasia. Some cats with aplastic anemia can have prolonged survival without resolution of the pancytopenia.     

Flow cytometric analysis of T-lymphocyte subsets in cats.

We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.     

Multiple myeloma in 16 cats: a retrospective study

BACKGROUND: There is limited published information regarding feline multiple myeloma. Diagnostic criteria are derived from canine studies and to our knowledge, have not been critically reviewed for cats. OBJECTIVE: To evaluate the clinical and laboratory findings in cats with multiple myeloma and appraise diagnostic criteria. METHODS: Retrospective evaluation of medical records was performed. Inclusion required an antemortem diagnosis of multiple myeloma using 2 of 4 criteria: 1) >or=20% plasma cells in the bone marrow, or >or=10% if atypical plasma cells; 2) paraproteinemia; 3) radiographically-evident osteolysis; 4) light chain proteinuria. Alternatively, a postmortem diagnosis was based on the findings of multiple plasma cell neoplasms, with marrow involvement. RESULTS: Sixteen cats were diagnosed with multiple myeloma between 1996 and 2004, with a median age of 14.0 years; 9 of 16 (56%) were castrated males, and 7 of 16 (44%) were spayed females. Laboratory abnormalities included hyperglobulinemia (14/16, 87.5%), with 11/14 (78.5%) monoclonal and 3/14 (21.4%) biclonal gammopathies; hypoalbuminemia (4/16, 25%); light chain proteinuria, (4/9, 44.4%); hypocholesterolemia (11/16, 68.7%); hypercalcemia, (3/15, 20%); nonregenerative anemia, (11/16, 68.7%); regenerative anemia, (1/16, 6.2%); neutropenia (5/15, 33.3%); thrombocytopenia (8/16, 50%); and marrow plasmacytosis (14/15, 93.3%). Plasma cells were markedly immature, atypical, or both in 10 of 12 (83.3%) cats. Focal or multifocal osteolysis was noted in 6 of 12 (50%) cats for which radiographs were available for review; generalized osteopenia was found in 1 (8.3%) cat. Noncutaneous, extramedullary tumors were found in all cats assessed, 7/7 (100%), including spleen (6), liver (3), and lymph nodes (4). The disease in 1 of 2 cats with cutaneous tumors progressed to plasmacytic leukemia. CONCLUSIONS: Common findings in feline multiple myeloma include atypical plasma cell morphology, hypocholesterolemia, anemia, bone lesions, and multi-organ involvement. Based on the results of this study, we advocate modifying diagnostic criteria in cats to include consideration of plasma cell morphology and visceral organ infiltration.     

Dysmyelopoiesis in the cat: a hematological disorder resembling refractory anemia with excess blasts in man.

Clinical and pathological findings in three cats affected with a myelodysplastic disorder are presented. This hematological disorder resembles that of refractory anemia with excess of blasts as seen in man. The hematological profile in man is one of peripheral cytopenia in one or all of the marrow cell lines which occurs despite a normal to hypercellular bone marrow. Quantitatively, the marrow has a preponderance of blasts (up to 20%). Qualitative abnormalities consist of dysthrombopoiesis and/or dyserythropoiesis and/or dysgranulopoiesis. Myelodysplastic disorders in the cat are a form of marrow failure often associated with infection with feline leukemia virus. The use of the term refractory anemia with excess of blasts appears to be applicable to the cat and should be considered in evaluating dysplastic disorders of the feline bone marrow.     

Determination of differential cell counts in feline bone marrow by use of flow cytometry

OBJECTIVE: To evaluate the potential usefulness of 2 flow cytometric methods for determination of differential cell counts in feline bone marrow. SAMPLE POPULATION: 10 bone marrow specimens from client-owned cats. PROCEDURE: Bone marrow specimens were stained with 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluated by use of flow cytometry. Differential counts were also determined by analysis of scatterplots of forward-angle versus side-angle light scatter of unstained specimens, obtained by use of flow cytometry (scatterplot method). Results of both flow cytometric methods were compared with differential cell counts determined by manually counting 1,000 cells on slides of Wright-stained smears. RESULTS: Staining with DiOC6 resulted in identification of mature and immature erythroid and myeloid cells and lymphocytes. Use of the scatterplot method resulted in identification of mature and immature erythroid and myeloid cells and metamyelocytes. However, to identify lymphocytes by use of the scatterplot method, bone marrow specimens were first labeled with an anti-major histocompatability class-II antibody. Comparison of results of the scatterplot method with manual counts yielded higher correlation coefficients and more similar mean values than did comparison of results of the DiOC6 method. Conclusions and Clinical Relevance: The scatterplot method provided more accurate and precise results than the DiOC6 method for determination of bone marrow differential cell counts in cats by use of flow cytometry. When combined with fluorescent labeling of lymphocytes, the scatterplot method has potential to provide rapid semiquantitative assessment of bone marrow differential cell counts in cats.     

Enhanced platelet reactivity in cats experimentally infected with feline infectious peritonitis virus

Platelet function was evaluated in six specific-pathogen-free cats prior to and following intraperitoneal inoculation with feline infectious peritonitis virus (FIPV). By 4 days post-inoculation, platelet samples from five of six cats responded with irreversible platelet aggregation to threshold concentrations of adenosine diphosphate (ADP). This was accompanied by enhanced platelet 14C-serotonin release (greater than 10%) in two cats. Compared to one of six baseline samples, five of five post-inoculation samples exhibited microaggregate formation in response to 20 microM epinephrine. Enhanced platelet 14C-serotonin release did not accompany these responses. Enhanced platelet responses to ADP and epinephrine were also observed on day 11 post-inoculation and day 16 (when one cat died) or 21 (the end of the study). Platelet 14C-serotonin release in response to 20 microM epinephrine increased markedly in three of five cats on day 21. Enhanced collagen-induced platelet responses were not demonstrated. Although the mechanism for the enhanced platelet responses observed on day 4 was unknown, a direct effect on the virus on platelets, mononuclear inflammatory cells, and endothelial cells must be considered.