Histopathology of experimental plague in cats.

Formalin-fixed paraffin-embedded archival tissues of seven adult cats of both sexes that died after being experimentally infected with Yersinia pestis were examined light microscopically to characterize the lesions. The cats were exposed in two groups using two routes of infection: ingestion of Y. pestis-infected rodent or a subcutaneous injection of Y. pestis to simulate a flea bite. Immunohistochemistry was performed on tissues from all organ systems from a representative cat from each group to determine the distribution of Y. pestis bacilli during infection. In all seven cats, bubonic plague lesions were seen. The lesions of pneumonic plague were present in two cats. Septicemic plague was confirmed in all seven cats by bacteriologic culture. Aggregations of bacteria were seen in lymphoid tissue in all cats and in lung tissues from the two cats with pneumonic plague. The most consistent histologic finding was necrosuppurative inflammation in the lymph nodes. Invariably, Y. pestis bacteria were present in large numbers at affected sites. Orally infected cats had more numerous lesions in the lymph nodes of the head and neck regions. These experimentally induced cases of feline plague document that cats are unique among carnivores in exhibiting bubonic, pneumonic, and septicemic plague following exposure to Y. pestis. The lesions of the orally infected cats were consistent with those previously described for naturally occurring Y. pestis infections in cats and corroborate the contention that cats most commonly contract plague by eating Y. pestis-infected rodents and not via flea bite. The histopathology of Y. pestis disease in these cats is comparable to that described for human plague.

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.

FeLV envelope protein (gp70) variable region 5 causes alterations in calcium homeostasis and toxicity of neurons.

In humans and animals, retroviruses have been implicated in nervous system disease. Our objective was to characterize the neurotoxicity of a peptide sequence derived from an animal retrovirus, the feline leukemia virus (FeLV). Using a peptide sequence from the subtype FeLV-C envelope protein variable region 5 (VR5), cytotoxicity was demonstrated in studies that evaluated neuronal survival, neurite outgrowth, and alterations in intracellular calcium ion concentration. The FeLV subtype isolate FeLV-CSarma possesses an envelope protein VR5 amino acid sequence that varies by four amino acids from the VR5 amino acid sequence of subtype FeLV-AGlasgow. The polypeptide representing the VR5 of FeLV-CSarma (FeLV-CVR5) is significantly more neurotoxic than the polypeptide sequence representing the VR5 of FeLV-AGlasgow (FeLV-AVR5). FeLV-CVR5 (> or = 3 microM) exposure resulted in significant dose-dependent neurotoxicity. Antibodies to FeLV-CVR5 blocked this effect. Neurite outgrowth was significantly reduced at all tested concentrations (3-12 microM) of FeLV-CVR5, with a 92% reduction in neurite length at 12 microM. FeLV-AVR5 was significantly less neurotoxic with respect to neurite outgrowth than was FeLV-CVR5. The significant reduction in neurotoxicity for FeLV-AVR5 illustrates the importance of the 4-amino-acid difference between it and FeLV-CVR5. Alterations in intracellular calcium ion concentration were associated with this neurotoxicity.

Allogeneic marrow transplantation in a cat with acute myeloid leukemia.

A listless, anorectic 2-year-old cat was found to have a normocytic, nonregenerative anemia. A diagnosis of acute myeloid leukemia type M6Er was made. Because the cat was young, had negative FeLV and feline immunodeficiency virus test results, had a sibling that could be used as a bone marrow donor, had received only 1 transfusion, and was in good health other than being anemic, allogeneic bone marrow transplantation (BMT) was attempted using marrow from the cat's sibling. The most life-threatening complication following BMT was hemorrhage caused by severe thrombocytopenia. Complete hematopoietic engraftment was evident 36 days after BMT. The cat then was discharged to its owner. In the first year, the cat continued to improve with the exception of an intractable dermatophytosis, which resolved eventually. The cat continues to do well 4 years after BMT. To our knowledge, this represents the first report of successful allogeneic BMT for a cat with acute myeloid leukemia.

Hyperthermic therapy for HIV infection.

The objective of this paper is to review what is known about the antiviral effects of fever and to highlight the scientific evidence supporting the hypothesis that hyperthermic therapy may prove to be a beneficial treatment modality for persons infected with HIV. Our hyperthermic hypothesis is based upon the mutant escape, quasispecies theory of HIV antigenic diversity. We propose that, if initiated during the asymptomatic stage of HIV infection, hyperthermia may prove to decrease the number of mutant HIV strains arising due to evolutionary pressures created by the patient's immune system, with a resultant prolongation of the asymptomatic period of infection. A review of the literature from three areas of investigation: the immune response to fever, heat as a tumor killing agent, and preliminary studies with fever and retroviral infections, strongly suggests that there is a good scientific basis for the use of hyperthermic therapy in a multimodal treatment approach to HIV infection.

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.

Effect of feline immunodeficiency virus infection on Toxoplasma gondii-specific humoral and cell-mediated immune responses of cats with serologic evidence of toxoplasmosis.

Serum samples from 89 cats with serologic evidence of toxoplasmosis were identified by using an enzyme-linked immunosorbent assay (ELISA) that detected Toxoplasma gondii-specific immunoglobulin M (IgM) or T. gondii-specific immunoglobulin G (IgG). Concurrent feline immunodeficiency virus (FIV) infection was detected in 36 cats using an ELISA for detection of FIV-specific IgG. The majority of the cats in both the FIV-seropositive and FIV-seronegative groups were male and > 5 years of age. FIV-seropositive cats were more likely to have T. gondii IgM titers without IgG (P < 0.05) or any T. gondii IgM titer (P < 0.05) than were FIV-seronegative cats. FIV-seronegative cats (1328) had a higher T. gondii IgG geometric mean titer than did FIV-seropositive cats (724) and were more likely to have T. gondii IgG titers > 1:2048 than were FIV-seropositive cats (P < 0.05). Cats with serologic evidence of both T. gondii and FIV infections had persistent T. gondii IgM titers for > 12 weeks. Lymphoblast transformation in response to concanavalin A, T. gondii-specific intracellular antigens, and T. gondii-specific secretory antigens was compared in T. gondii seropositive and FIV-seronegative cats, cats with serologic evidence of T. gondii infection alone, and cats with serologic evidence of concurrent FIV and T. gondii infections. Lymphocytes from all but one cat in the FIV-seropositive group responded to concanavalin A. Whereas lymphocytes from FIV-seronegative cats with serologic evidence of toxoplasmosis responded to T. gondii-specific antigens, four of five of the FIV-seropositive cats with concurrent serologic evidence of toxoplasmosis did not.

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.

Effect of primary phase feline immunodeficiency virus infection on cats with chronic toxoplasmosis.

The effect of primary phase feline immunodeficiency virus (FIV) infection on clinical signs, hematological values, Toxoplasma gondii oocyst shedding, T. gondii-specific serology, T. gondii-specific cell-mediated immune responses, non-specific cell-mediated immune responses, and lymphocyte subpopulations from cats with experimentally induced chronic toxoplasmosis was studied. No significant clinical or hematologic abnormalities were noted following inoculation with FIV. T. gondii-specific IgM was significantly increased, concanavalin A, T. gondii tachyzoite antigen and T. gondii secretory antigen induction of lymphocyte transformation were significantly suppressed, and CD4+ cell numbers were significantly decreased following inoculation with FIV. The changes were attributed to FIV effects on the immune system and resultant activated toxoplasmosis.

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.

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)

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.

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.

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.

Platelet aggregation and ATP secretion in whole blood of normal cats and cats homozygous and heterozygous for Chediak-Higashi syndrome.

A rapid and simple technique using the Whole Blood Lumi-Aggregometer was used to study storage pool disease in Chediak-Higashi homozygote and heterozygote cats. Feline Chedlak-Higashi platelets aggregated after the addition of both ADP and collagen. During platelet aggregation, ATP secretion was assayed; the whole blood aggregometer is effective in detecting decreased levels of secretable ATP in homozygote cats. No storage pool deficiency was found in heterozygote cats. However, upon analysis of impedance tracings, a decreased platelet aggregation response was seen in both homozygote and heterozygote cats. These results suggest that prolonged bleeding times in Chediak-Higashi cats may involve a mechanism in addition to a dense granule deficiency.

Experimental transmission and pathogenesis of immunodeficiency syndrome in cats.

We describe the identification, experimental transmission, and pathogenesis of a naturally occurring powerfully immunosuppressive isolate of feline leukemia virus (designated here as FeLV-FAIDS) which induces fatal acquired immunodeficiency syndrome (AIDS) in 100% (25 of 25) of persistently viremic experimentally infected specific pathogen-free (SPF) cats after predictable survival periods ranging from less than 3 months (acute immunodeficiency syndrome) to greater than one year (chronic immunodeficiency syndrome), depending on the age of the cat at time of virus exposure. The pathogenesis of FeLV-FAIDS-induced feline immunodeficiency disease is characterized by: a prodromal period of largely asymptomatic viremia; progressive weight loss, lymphoid hyperplasia associated with viral replication in lymphoid follicles, lymphoid depletion associated with extinction of viral replication in lymphoid follicles, intractable diarrhea associated with necrosis of intestinal crypt epithelium, lymphopenia, suppressed lymphocyte blastogenesis, impaired cutaneous allograft rejection, hypogammaglobulinemia, and opportunistic infections such as bacterial respiratory disease and necrotizing stomatitis. The clinical onset of immunodeficiency syndrome correlates with the replication of a specific FeLV-FAIDS viral variant, detected principally as unintegrated viral DNA, in bone marrow, lymphoid tissues, and intestine. Two of seven cats with chronic immunodeficiency disease that survived greater than 1 year after inoculation developed lymphoma affecting the marrow, intestine, spleen, and mesenteric nodes. Experimentally induced feline immunodeficiency syndrome, therefore, is a rapid and consistent in vivo model for prospective studies of the viral genetic determinants, pathogenesis, prevention, and therapy of retrovirus-induced immunodeficiency disease.

Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma.

We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week-old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S.

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.