Clonality analysis of various hematopoietic disorders in cats naturally infected with feline leukemia virus

The clonality analysis of the bone marrow cells was carried out by detecting the integrated proviruses of feline leukemia virus (FeLV) to understand the pathogenesis of FeLV-associated hematopoietic disorders in cats. Bone marrow cells from 4 cases with acute myeloid leukemia (AML), 9 cases with myelodysplastic syndromes (MDS), 2 cases with pure red cell aplasia (PRCA) and 3 healthy carriers infected with FeLV were subjected to Southern blot analyses using an exogenous FeLV probe. Clonal hematopoiesis was found in all the cases with AML and in 6 of the 9 cases with MDS, but not in the cases with both PRCA and healthy carriers infected with FeLV. In the 2 cases with MDS, it was thought that the same clones of the hematopoietic cells might proliferate before and after the progression of the disease irrespective of the changes of the hematological diagnoses by cytological examination. This study indicates that MDS in cats is a disease manifestation as a result of clonal proliferation of hematopoietic cells and can be recognized as a pre-leukemic state of AML.     

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.     

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.     

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.     

Clinical aspects of feline immunodeficiency and feline leukemia virus infection

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are retroviruses with a global impact on the health of domestic cats. The two viruses differ in their potential to cause disease. FIV can cause an acquired immunodeficiency syndrome that increases the risk of developing opportunistic infections, neurological diseases, and tumors. In most naturally infected cats, however, FIV itself does not cause severe clinical signs, and FIV-infected cats may live many years without any health problems. FeLV is more pathogenic, and was long considered to be responsible for more clinical syndromes than any other agent in cats. FeLV can cause tumors (mainly lymphoma), bone marrow suppression syndromes (mainly anemia) and lead to secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. Today, FeLV is less important as a deadly infectious agent as in the last 20 years prevalence has been decreasing in most countries.     

Chronic myelomonocytic leukemia in a cat

A 1-year-old spayed domestic short-haired cat was referred with anorexia and weight loss. Hematologic findings indicated nonregenerative anemia, severe neutropenia and monocytosis. The feline leukemia virus (FeLV) antigen test was positive reaction by enzyme-linked immunosorbent assay. Dysgranulopoiesis with slight increase in blast cells were observed in bone marrow smears. On the basis of blood and bone marrow findings, the cat was diagnosed as chronic myelomonocytic leukemia (CMMoL), which possibly corresponds to a kind of the subtypes in human myelodysplastic syndrome (MDS).     

Expression of viral proteins in feline leukemia virus-associated enteritis

Fourteen cases of feline leukemia virus (FeLV)-associated enteritis were immunohistologically examined for the expression of FeLV proteins gp70, p27, and p15E in the jejunum, mesenteric lymph nodes, spleen, and bone marrow. Results were compared with those of FeLV-infected cats without intestinal alterations. Other viral infections and specific bacterial, fungal, and parasitic infections were excluded by standard microbiologic methods, histopathology, immunohistology, and in situ hybridization. In FeLV-associated enteritis, FeLV gp70 and p15E were strongly expressed in intestinal crypt epithelial cells. In contrast, FeLV-positive cats without intestinal alterations showed only faint staining for gp70 and p15E and comparatively strong p27 expression in these cells. Findings suggest a direct relation between FeLV infection and alterations in intestinal crypt epithelial cells that may be attributed to the envelope proteins gp70 and p15E and/or their precursor protein. Distinct similarities to the intestinal changes in the experimentally induced FeLV-feline AIDS syndrome are obvious, suggesting that naturally occurring feline AIDS variants may be responsible for FeLV-associated enteritis.     

Influence of thymectomy on the susceptibility of cats to feline leukemia virus and lymphosarcoma.

Twelve cats were thymectomized at 5 weeks of age. Six of these cats were inoculated at 8 weeks of age and 6 at 4 months of age with the Rickard (R) strain of feline leukemia virus (FeLV), which produces a high incidence of thymic lymphosarcoma. Two groups of age-matched nonthymectomized cats were inoculated with the same FeLV-R stock. Thymectomy prior to FeLV infection had no influence on the induction of viremia or the incidence of lymphosarcoma. In the FeLV-inoculated nonthymectomized cats, lymphosarcoma developed in the thymus. In the thymectomized cats, lymphosarcoma developed in the intestine, mesenteric lymph nodes, and bone marrow, but the malignant lymphoblasts had surface markers characteristic of feline T lymphocytes. It was concluded that the presence of the thymus per se is not required for infection and oncogenesis by FeLV and that feline T lymphocytes may be transformed after peripheralization to other tissues.     

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.     

Comparative examination of cats with feline leukemia virus-associated enteritis and other relevant forms of feline enteritis

Cats with feline leukemia virus (FeLV)-associated enteritis (FAE), enteritis of other known viral etiology (parvovirus [PV], enteric coronavirus [CoV]), and enteritis of unknown etiology with histologic features similar to those of FAE and PV enteritis (EUE) and FeLV-negative and FeLV-positive cats without enterocyte alterations were examined. Amount and types of infiltrating leukocytes in the jejunum and activity and cellular constituents of mesenteric lymph nodes, spleen, and bone marrow were determined. PV and CoV infections were confirmed by immunohistologic demonstration of PV and CoV antigen, ultrastructural demonstration of viral particles in the intestinal content, and in situ hybridization for PV genome. FeLV infection was detected by immunohistology for gp70, p27, and p15E. Latent FeLV infection was excluded by polymerase chain reaction methods for exogenous FeLV DNA. Enterocyte lesions involved the crypts in cats with PV enteritis, FAE, and EUE and the villous tips in cats with CoV enteritis. Inflammatory infiltration was generally dominated by mononuclear cells and was moderate in the unaltered intestine and in cats with PV enteritis and marked in cats with FAE, CoV enteritis, and EUE. In cats with EUE, myeloid/histiocyte antigen-positive macrophages were relatively numerous, suggesting recruitment of peripheral blood monocytes. Lymphoid tissues were depleted in cats with PV enteritis and with EUE but were normal or hyperplastic in cats with FAE. Bone marrow activity was decreased in cats with PV enteritis; in cats with FAE or EUE and in FeLV-positive cats without enterocyte alterations, activity was slightly increased. In cats with FAE and PV enteritis, a T-cell-dominated response prevailed. EUE showed some parallels to human inflammatory bowel disease, indicating a potential harmful effect of infiltrating macrophages on the intestinal epithelium.     

The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma

Ocular sarcoma was diagnosed by light microscopic examination in enucleated globes ( n  = 4), orbital tissue biopsy ( n  = 1) and ocular evisceration contents ( n  = 1) from six cats. To determine if feline leukemia virus (FeLV) or a replication-defective FeLV, feline sarcoma virus (FeSV), was present in these ocular sarcomas, immunohistochemistry (IHC) and polymerase chain reaction (PCR) for FeLV were utilized. Immunohistochemical staining for FeLV glycoprotein 70 (gp70) was performed on all six formalin-fixed, paraffin-embedded tumors using an avidin–biotin complex technique. DNA was extracted from each specimen and a 166 bp region of the FeLV long-terminal repeat (LTR) was amplified by PCR. All tumors were composed primarily of spindle cells; two neoplasms had PAS-positive basement membrane enveloping areas of spindle cells. All tumors involved the uvea and five of six tumors showed transcleral extension, one of which invaded the optic nerve. Immunohistochemical staining for FeLV gp 70 was negative. PCR to amplify a portion of the FeLV LTR was negative. Based on these findings of these limited number of cases, FeLV/FeSV may not play a role in the tumorigenesis of feline ocular sarcomas. However, additional tumors representing all morphological subtypes should be investigated for the presence of viral antigen and DNA. It is important to determine the etiology and pathogenesis of these malignant ocular sarcomas. If the cell of origin and pathogenesis involve ocular and lenticular injury, and FeLV/FeSV is not present, then the clinical management of cases of feline ocular trauma, uveitis and glaucoma may prevent the development of this tumor.     

Hematologic abnormalities preceding apparent recovery from feline leukemia virus infection

A 5-month-old domestic short-haired cat was referred to the Western College of Veterinary Medicine because of hemorrhage from the mouth. Neutropenia and bone marrow dyscrasia developed within 2 weeks, and results of a fluorescent antibody test for feline leukemia virus (FeLV) were positive. During the next 6 months, the feline oncornavirus cell membrane-associated antibody titer increased, the hematologic abnormalities regressed, and the cat became negative to the FeLV fluorescent antibody test (FeLV test). The results of 3 consecutive FeLV tests were negative, and the cat has been clinically normal for the past 18 months.     

Serological survey of Toxoplasma gondii,feline immunodeficiency virus and feline leukaemia virus in urban stray cats in Belgium

Three hundred and forty-six serum samples taken between 1998 and 2000 from urban stray cats in the city of Ghent were tested for antibodies to Toxoplasma gondii and feline immunodeficiency virus (FIV), and antigens of feline leukemia virus (FeLV). Of these 346 samples, 243 (70.2 per cent) were seropositive for Tgondii. Thirty-nine cats (11.3 per cent) had antibodies against FIV and 13 (3.8 per cent) had circulating antigens of FeLV. Fewer of the female cats had FIV and heavier cats had a higher seroprevalence of FIV. Exact logistic regression showed that cats that were infected with FIV were more likely to be infected with T gondii (P = 0.04), and the cats with FIV had a higher titre of Tgondii antibodies than FIV-negative animals. However, FeLV was not associated with either T gondii or FIV.     

Acute myeloid leukemia with basophilic differentiation (AML,M-2B) in a cat

A 4-year-old, neutered male Domestic Shorthair cat with a history of depression, anorexia, and weight loss was diagnosed with acute myelogenous leukemia (AML). The cat tested positive by both the feline immunodeficiency virus antibody test and feline leukemia virus enzyme-linked immunosorbent assay test. Results of cytochemical stains on peripheral blood and bone marrow specimens indicated acute myeloid leukemia with unusual basophilic differentiation (AML, M-2B).     

Suspected myelodysplastic/myeloproliferative neoplasm in a feline leukemia virus‐negative cat

A 10‐year‐old castrated Domestic Short‐Haired cat was presented to a primary care veterinarian for a wellness examination and laboratory examination for monitoring of diabetes mellitus. The CBC revealed marked thrombocytosis, leukopenia and macrocytic, normochromic anemia. The cat tested negative for FeLV and feline immunodeficiency virus, but was positive for Mycoplasma haemominutum by PCR. Hematologic abnormalities were not responsive to therapy, so a repeat CBC and a bone marrow aspiration for cytology were performed. Additional blood smear findings included anisocytosis with megaloblastic erythroid precursors, large platelets, eosinophilic myelocytes and metamyelocytes, and rare unidentified blasts. The bone marrow smear was highly cellular, and the cytologic pattern was consistent with myelodysplastic syndrome with an erythroid predominance. At that time, 15% blasts were present. The cat was treated with a vitamin K₂ analog, doxycycline, and prednisolone, but without a clinical response. Within 3 months, euthanasia was elected due to declining quality of life, and a necropsy was performed. Postmortem bone marrow smears were highly cellular and dominated by monomorphic blasts of unknown line of origin (52%), persistent marked erythroid and megakaryocytic dysplasia, and ineffective erythropoiesis and granulopoiesis. Immunohistochemical, immunocytochemical, and cytochemical stains resulted in a diagnosis of acute myeloid leukemia of unclassified type. Additional histologic findings included mixed hepatitis with trematode infestation and lymphoplasmacytic interstitial nephritis with fibrosis. The marked thrombocytosis with myelodysplastic syndrome and the FeLV‐negative status of this cat were unusual. The difficulty in classifying the myelodysplasia and subsequent leukemia highlights a need for further reporting and characterization of these types of disease.     

Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection

In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection.     

Selective impairment of humoral immunity in feline leukemia virus-induced immunodeficiency.

We used a panel of in vitro assays to investigate the nature of immune dysfunction in cats infected with FeLV-FAIDS, a naturally occurring, molecularly cloned feline leukemia virus (FeLV) isolate which induces a fatal immunodeficiency syndrome in infected cats. During the asymptomatic period preceding immunodeficiency disease, we were unable to detect any deficits in concanavalin A-induced blastogenesis, xenogeneic mixed-lymphocyte reaction assays, stimulation of lymphocytes by soluble protein antigen, and cytotoxic T lymphocyte assays. However, during this period humoral immune responses in the FeLV-FAIDS-infected cats were dramatically impaired. As early as 9 weeks after virus inoculation, the ability to mount either an IgM or IgG response to soluble protein antigens was lost. Neither B cell function, as assessed by lipopolysaccharide-induced blastogenesis or circulating B cell numbers, as assessed by immunofluorescence, differed between infected and control cats. These results suggest that FeLV-FAIDS infection may impair a subpopulation of T helper cells, that provides help for the production of antibody. Consistent with earlier observations of cats naturally infected with FeLV, our results indicate that early impairment of humoral immunity is an important component of the immunodeficiency syndrome induced by FeLV in cats.     

Real-time PCR investigation of feline leukemia virus proviral and viral RNA loads in leukocyte subsets

Cats exposed to feline leukemia virus (FeLV), a naturally occurring gammaretrovirus develop either progressive or regressive infection. Recent studies using analyses with enhanced sensitivity have correlated loads throughout FeLV with the clinical outcome, though remarkably, during the acute phase of infection, proviral and viral RNA burdens in the peripheral blood do not differ between groups. We hypothesized that viral loads in specific leukocyte subsets influence the infection outcome. Using a method established to determine the proviral and cell-associated viral RNA loads in specific leukocyte subsets, we evaluated viral loads in eleven FeLV-exposed specific pathogen-free (SPF) cats 2.5 years post-infection. Six cats had undergone regressive infection whereas five were persistently viremic. Aviremic cats had lower total proviral blood loads than the persistently infected cats and FeLV proviral DNA was shown to be integrated into genomic DNA in four out of four animals. Lymphocytes were predominantly infected vs. moncytes and granulocytes in aviremic cats. In contrast, persistently viremic cats were provirus-positive in all leukocyte subsets. The acute phase kinetics of FeLV infection were analyzed in two additional cats; an early lymphoreticular phase with productive infection in lymphocytes in both cats and in monocytes in one cat was followed by infection of the granulocytes; both cats became persistently infected. These results indicate that FeLV persistent viremia is associated with secondary viremia of bone marrow origin, whereas regressive cats only sustain a non-productive infection in low numbers of lymphocytes.     

Steroid-responsive neutropenia in a cat with progressive feline leukemia virus infection

An 8-month-old female domestic shorthair cat was presented to the Animal Medical Center with anorexia, lethargy, and mild gastrointestinal signs. A CBC revealed a profound neutropenia, and serologic testing with an in-house test kit (SNAP FIV/FeLV Combo, IDEXX) was positive for feline leukemia virus (FeLV) antigen. Serial hematologic examinations during hospitalization showed a persistent neutropenia with occasionally severe anemia and thrombocytopenia. Prednisolone administration afforded complete hematologic remission within 3 days. Four weeks after the premature discontinuation of prednisolone, the patient relapsed; however, complete and prolonged hematologic remission was achieved after prednisolone was re-induced. Bone marrow aspiration cytology was consistent with immune-mediated destruction of the mature myeloid cells. steroid-responsive (likely immune-mediated) cytopenias rarely occur in cats with progressive FeLV infection. Although only a few cases of FeLV-positive, severely neutropenic cats that responded to immunosuppressive therapy have been reported, this case highlights that a grave prognosis should not always be given to these FeLV-positive cats.     

Safety and efficacy studies of live- and killed-feline leukemia virus vaccines.

The safety and the efficacy of several feline leukemia virus (FeLV) vaccines for 16-week-old kittens were determined. Vaccines were derived from an FL74 lymphoblastoid cell line that has been in continuous tissue culture passage for about 4 years. The vaccines were made from living virus, formaldehyde-inactivated whole FL74 cells, and formaldehyde-inactivated whole virus. The efficacy of each produced vaccine was determined by challenge exposure of vaccinated cats with virulent FeLV. The two formaldehyde-inactivated vaccines were found to be safe for use in kittens. Neither vaccine produce a significant feline oncornavirus-associated cell membrane antigen or virus-neutralizing antibody response, nor did they prevent infection with virulent FeLV. The inactivated whole-virus vaccine, however, did substantially decrease the proportion of kittens infected with virulent FeLV that became persistently viremic. In contrast, the whole FL74 cell vaccine did not reduce the number of infected kittens that became persistently viremic. The live-virus vaccine was found to be both safe and efficacious. About a half of the kittens vaccinated with live virus had transient bone marrow infection that lasted from 2 to 4 weeks. Viral antigen was not detected in peripheral blood, and infective virus was not shed in saliva, urine, or feces during the period that the vaccinal virus could be recovered from the bone marrow. In addition, there was no horizontal spread of vaccinal virus from vaccinated to non-vaccinated cagemates. Within several weeks, vaccinated kittens demonstrated no clinical or hematologic abnormalities and had high serum levels of feline oncornavirus-associated cell membrane antigen and virus-neutralizing antibody. Kittens vaccinated with living FeLV were resistant to infection with virulent virus.