Influence of antibody infusion on pathogenesis of experimental feline leukemia virus infection.

For examination of the influence of antibody on the pathogenesis of feline leukemia virus (FeLV) infection, 12 weanling specific-pathogen-free cats were inoculated with isolates of FeLV and were treated beginning at 7, 19, 21, 24, 34, or 49 days post inoculation (DPI) with feline anti-FeLV hyperimmune serum (10 infusions, 37 mg globulin/kg each at 48-hr intervals). Anti-FeLV serum infusion initiated at 7 DPI prevented the onset of hematopoietic cell infection and viremia. Antibody treatment initiated at 19 or 24 DPI abrogated recently established FeLV viremia and extinguished p27 expression in bone marrow and blood cells. Viremia established for longer periods was refractory to antibody infusion despite establishment of enzyme-linked immunosorbent assay antibody titers of 1:80 to 1:320 in the treated cats. Latent FeLV infection was a sequel to antibody-induced curtailment of viral replication in bone marrow cells and was able to reactivate spontaneously in vivo as well as in vitro.

In vitro erythrocytopathic activity of an aplastic anemia-inducing feline retrovirus.

Although feline leukemia viruses (FeLV) cause a spectrum of proliferative and anti-proliferative diseases in vivo, in vitro studies demonstrating cell lineage-specific pathogenic properties of feline retroviruses have been rare. We describe here an efficient in vitro system that demonstrates the selective cytopathic effect of a molecularly cloned anemogenic FeLV (FeLV-Sarma-subgroup C; FSC) on erythroid progenitor cells. Forty-eight-hour coculture of normal feline bone marrow mononuclear cells with an underlayer of FSC-infected feline fibroblasts (FeF) resulted in infection of 60% to 90% of marrow mononuclear cells and pronounced depletion of early erythroid progenitor cells (BFUe). The dramatic depletion of BFUe was specific for FSC and did not occur in marrow cells infected with a molecularly cloned nonanemogenic subgroup A FeLV (FeLV 1161E; F6A). The ablation of BFUe by FSC in vitro paralleled both the decrease in BFUe and the induction of aplastic anemia in vivo. This combination of marrow cell infection by coculture and colony-forming unit (CFU) assessment by methylcellulose assay provides a reliable in vitro technique for studies of mechanisms involved in retrovirus-induced marrow aplasias.

Effect of recombinant human granulocyte colony-stimulating factor on hematopoiesis in normal cats.

The objective of this study was to determine how recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects hematopoiesis in normal cats. Recombinant human G-CSF was given at 3.0, 5.0, and 10.0 micrograms/kg to two cats each s.c. twice daily for 21 days. This resulted in significant (p less than 0.01) elevations of peripheral blood neutrophils from 3.0- to 9.2-fold above pretreatment levels and significantly (p less than 0.02) above levels of nontreated control cats (n = 4). A statistically significant dose-related response was not seen at these dosages in any parameter evaluated. The period of maximum neutrophilia occurred between days 10 and 14 of rhG-CSF treatment, with maximum neutrophil counts ranging from 20,370 cells/microliters to 61,400 cells/microliters (normal is less than 12,500). Lymphocytosis (greater than 7000 lymphocytes/microliters) and monocytosis (greater than 850 monocytes/microliters) were observed in 50% of the cats receiving rhG-CSF during the period of maximal neutrophil stimulation. Monocyte counts in treated cats were significantly (p less than 0.01) elevated over those of treatment controls on days 12-17. Lymphocyte numbers in rhG-CSF-treated cats were significantly elevated (p less than 0.05) over pretreatment controls on days 12 and 14 of rhG-CSF treatment. No significant changes were observed in reticulocyte counts, platelet counts, or hematocrit levels. By day 19, neutrophil levels had dropped significantly (p less than 0.01) from the maximum neutrophil levels, with one cat attaining a normal blood neutrophil count by day 21 of rhG-CSF treatment. Marrow aspirates revealed an overall increase in marrow cellularity through day 14 of treatment in rhG-CSF-treated cats, with increased myeloid:erythroid ratios (two- to ninefold) over those of nontreated controls. The erythroid and lymphoid component of the marrow decreased from day 0 to day 14, whereas the early myeloid progenitors (myeloblasts, progranulocytes, and myelocytes) increased significantly (p less than 0.05). No significant differences in the percentage of later myeloid forms in the marrow were observed over the treatment period. In vitro colony-forming assays of marrow obtained from treated cats revealed increases in granulocyte-macrophage colony-forming units (CFU-GM) through day 14, with subsequent decreases by day 21 of rhG-CSF treatment. Recombinant human G-CSF was also effective at in vitro stimulation of feline marrow cells from untreated cats in a dilution study, with maximal CFU-GM formation at 0.1 microgram rhG-CSF/ml assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Neutrophil function in normal and Chediak-Higashi syndrome cats following administration of recombinant canine granulocyte colony-stimulating factor.

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) on leukocyte counts and neutrophil function in clinically normal cats and in cats heterozygotic and homozygotic for Chediak-Higashi syndrome (CHS) were examined. CHS is a genetic disease characterized by neutropenic episodes and defects in a variety of phagocyte functions. Short-term administration of rcG-CSF at 10 micrograms/kg body weight resulted in a five- to tenfold increase in circulating granulocytes by day 10 of administration and normalizes CHS neutrophil counts by day 3. The drug was specific for neutrophils as determined by differential cell counts. Neutrophil chemotaxis under agarose and phagocytosis of Escherichia coli were characterized following administration of rcG-CSF in vivo. Granulocytes elicited by rcG-CSF show enhanced chemotactic abilities toward activated serum, increased spontaneous migration, and an enhanced ability to ingest opsonized E. coli. At a concentration of 1 nM rcG-CSF in vitro, chemotaxis and spontaneous migration were increased, with no effect on phagocytosis. CHS neutrophil function was improved by administration of rcG-CSF in all parameters studied, although the defect in chemotaxis was present throughout the treatment period. We conclude from this study that neutrophils elicited by rcG-CSF are functionally enhanced and that rcG-CSF may be a viable therapy for CHS and other related disorders.

Characterization of osteopenia in feline mucopolysaccharidosis VI and evaluation of bone marrow transplantation therapy.

Studies on a feline model of MPS VI demonstrated a marked osteopenia in iliac crest bone samples from young adult animals with fewer, finer trabeculae. In the absence of significant differences in bone remodeling, this was considered due to defects in endochondral ossification and the formation of fewer trabeculae. Cell-level bone formation was normal despite the presence of vacuolated osteoblasts. Affected animals had vacuolated osteocytes in larger lacunae. Cats of the same age who had received a bone marrow transplant 12 months prior as young kittens, had significantly more trabecular bone with thicker trabeculae. The presence of smaller osteocyte lacunae in these animals as compared to their untreated MPS VI cats appeared to be a direct effect of bone marrow transplantation and a useful parameter to monitor its efficacy.

Induction of aplastic anemia by intra-bone marrow inoculation of a molecularly cloned feline retrovirus.

Intra-bone marrow inoculation of cells infected with molecularly cloned feline retrovirus (FeLV-C-Sarma [FSC]) associated with aplastic anemia was examined to test the hypothesis that cell-to-cell transmission of virus might facilitate marrow cell infection and anemogenesis, a possibility suggested by in-vitro co-culture experiments. IBM inoculation of either FSC-infected feline marrow cells or fibroblasts of weanling cats bypassed age-related restriction of FSC replication, initiated viremia, caused irreversible depletion of erythroid burst forming units, and induced rapid fatal aplastic anemia. A second significant finding observed with FSC infection was pronounced systemic lymphoid depletion. The direct bone marrow inoculation system described facilitates experimental study of retrovirus-target cell interactions involved in erythroid aplasia.

Infectious feline leukaemia virus is erythrosuppressive in vitro.

The direct effect of the feline leukaemia virus (FeLV) on erythroid colony formation in vitro was investigated. Bone marrow mononuclear cells (BMMC) from FeLV-naïve, specific-pathogen-free (SPF), adult cats were inoculated with FeLVs of characterized strains and biologically cloned subgroups and the subsequent development of colony forming units-erythroid (CFUE) and burst forming units-erythroid (BFUE) and colony forming units-granulocyte-macrophage (CFUGM) was monitored. Exposure to the anaemia-causing Kawakami-Theilen strain of FeLV (FeLV-KT), a phenotypic mixture of subgroups A, B, and C, caused constant depression of day 2 CFUE (to 47% of sham-inoculated controls), day 4 CFUE (41% of controls), and day 10 BFUE (38% of controls). CFUGM were unaffected. The lymphoma-causing Rickard strain of FeLV (FeLV-R-TL) caused sporadic depression of CFUE and BFUE. In contrast, neither FeLV-R passaged through feline embryonic kidney fibroblasts (FeLV-R-CRFK) nor biologically cloned, subgroup-specific, FeLVs of fibroblast origin, caused decrements in CFUE or BFUE, suggesting that fibroblast passage attenuated the direct erythrosuppressive effect of FeLV. Suppression of CFUE and BFUE by lymphoma cell-origin FeLV was dependent on infectious virus and was associated with FeLV replication by the cultured myelomonocytic precursor cells. Attenuation of infectivity by heat or u.v. restored CFUE and BFUE development. Examination of the relationship between viral infectivity (VI), viral protein concentration, and CFUE suppression showed that the infectious FeLV was 20-fold more effective than u.v.-inactivated FeLV as an inhibitor of erythrogenesis in vitro.

Restoration of neutrophil and platelet function in feline Chediak-Higashi syndrome by bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) was successfully performed in four Chediak-Higashi (CHS) syndrome affected cats. Preparatory regimens included selective intestinal flora decontamination, fractionated total body irradiation for myeloablation, and prophylactic treatment for graft-versus-host disease with cyclosporin A. Neutrophil chemotaxis under-agarose and whole-blood platelet aggregation/secretion were characterized prior to BMT and after engraftment of donor-origin marrow cells. Liver and kidney biopsies were obtained and evaluated by light and electron microscopy before, and at 6 months post-BMT to determine what effect BMT might have on abnormal lysosome fusion in hepatocytes and renal tubule cells. The platelet storage pool defect was resolved by day 40 post-BMT. In vitro neutrophil migration in all cats appeared to improve with time after BMT and complete restoration was evident by day 175 post-BMT. No apparent differences were evident in either the liver or the kidney at 6 months post-BMT. One cat developed seizures and one developed posterior paresis 5 months post-BMT; neurologic impairment ultimately resulted in death of two cats at 6 and 8 months post-BMT, respectively. Neurologic lesions in both cats were characterized by non-suppurative encephalitis. Allogeneic BMT successfully corrected the neutrophil migration defect and platelet storage pool deficiency but had no effect on lysosome distribution in liver and kidney cells of CHS cats.

Urine glycosaminoglycan concentrations in mucopolysaccharidosis VI-affected cats following bone marrow transplantation or leukocyte infusion.

Urinary glycosaminoglycan (GAG) concentrations were determined in nineteen normal cats (eleven kittens and eight adult cats), eighteen mucopolysaccharidosis VI (MPS VI)-affected untreated cats (ten kittens and eight adult cats), thirteen cats MPS VI-affected cats following bone marrow transplants (BMT), and two MPS VI-affected cats following intravenous infusion of leukocytes from normal cats. Mucopolysaccharidosis VI-affected cats treated with BMT had a precipitous decrease in urinary GAG by day 7 post-BMT, then a transient increase just prior to engraftment, followed by a sustained decrease to within, or near, the range of urinary GAG concentration established for normal cats. The pre-engraftment changes in urinary GAG excretion were reproduced by leukocyte infusion. After infusion of comparable members of normal peripheral blood leukocytes, a significant decrease in urinary GAG concentrations, specifically dermatan sulfate (DS), was seen with a nadir at day 5 post-infusion, followed by a return by day 9 to pre-infusion values. Post-engraftment, a continued low urinary GAG concentration with a specific decrease in DS can be utilized to document successful autologous engraftment in MPS VI-affected cats.

Plague (Yersinia pestis) in cats: description of experimentally induced disease.

Sixteen healthy cats were fed a 6-wk-old laboratory mouse that had died of experimentally induced Yersinia pestis infection (strain NM77-538), to simulate oral exposure to plague. The cats were closely monitored after ingestion. Physical exams were performed and vital signs were recorded daily. Plague antibody titers and cultures of blood, throat, and oral cavity were performed daily. Complete blood counts and biochemistry panels were performed every 3 d. Complete necropsies were performed on any cats that died. Cats exhibited one of three responses following ingestion of one plague-infected mouse; they either died (6/16 or 38%), developed transient illness and recovered (7/16 or 44%) or showed no signs of illness (3/16 or 19%). A continual fever greater > 40 degrees C was associated with a poor prognosis. The highest antibody titers developed in the group that shed the plague bacillus over an extended period of time. Blood, throat, and oral cavity cultures were positive in 100% of the fatal cases. Throat cultures were positive in 75% of the exposed cats. In contrast to other carnivores, cats infected with Y. pestis exhibit bubo formation and pneumonic lesions similar to those seen in people with plague. Because of the potential transmission of Y. pestis from cats to people, development of a plague vaccine for cats may be warranted.