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Prevalence of hematological abnormalities and detection of infected bone marrow cells in asymptomatic cats with feline immunodeficiency virus infection
Veterinary Microbiology 136 (2009) 217–225
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Prevalence of hematological abnormalities and detection of infected bone marrow cells in asymptomatic cats with feline immunodeficiency virus infection Yasuhito Fujino 1,*, Hiroshi Horiuchi 1, Fuminori Mizukoshi, Kenji Baba, Yuko Goto-Koshino, Koichi Ohno, Hajime Tsujimoto Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, the University of Tokyo, Tokyo 113-8657, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 June 2008 Received in revised form 31 October 2008 Accepted 4 November 2008
Peripheral blood cytopenia such as anemia, leukopenia with neutropenia and thrombocytopenia is frequently observed in cats infected with feline immunodeficiency virus (FIV). Although previous studies report that cytopenia has been observed in FIVinfected symptomatic cats, yet the asymptomatic cats also present cytopenia occasionally. In the present study, hematological and virological analyses in FIV-infected asymptomatic cats were carried out to understand the prevalence and pathogenesis of peripheral blood cytopenia in FIV infection. Hematological abnormalities were detected in 24 of 50 FIVinfected asymptomatic cats (48%) in which no other cause of cytopenia than FIV infection was observed. Anemia only, neutropenia only, thrombocytopenia only, bicytopenia and pancytopenia were observed in 10%, 10%, 6%, 14% and 8%, respectively. Bone marrow (BM) examination was performed in 8 FIV-infected asymptomatic cats with peripheral blood cytopenia. Myeloid dysplasia was observed in 4 cats with neutropenia of which 2 cats with concurrent thrombocytopenia presented morphological abnormalities of megakaryocytes. FIV-infected BM cells in the 8 cats were analyzed by PCR and immunocytochemistry. Lobulated mononuclear cells in BM were infected with FIV in 5 cats with neutropenia of which 2 cats with concurrent thrombocytopenia showed FIV-infected megakaryocytes. Parts of isolated stromal cells from BM were infected with FIV in all the 8 cats. Present results suggest that FIV infection of BM cells can cause peripheral blood cytopenia and myelodysplasia even if the cat is asymptomatic. Such FIV-related hematological abnormalities are supposed to be diagnosed as FIV-myelopathy. ß 2008 Published by Elsevier B.V.
Keywords: FIV Cat Peripheral blood cytopenia Myelodysplasia Stromal cells
1. Introduction Feline immunodeficiency virus (FIV) is a retrovirus, subfamily Lentivirinae, associated with acquired immu-
* Corresponding author at: Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: +81 3 5841 8004; fax: +81 3 5841 8178. E-mail address: [email protected] (Y. Fujino). 1 These authors contributed equally to this work. 0378-1135/$ – see front matter ß 2008 Published by Elsevier B.V. doi:10.1016/j.vetmic.2008.11.007
nodeficiency syndrome (AIDS) in cats (Pedersen et al., 1987, 1989). Cats infected with FIV may remain asymptomatic for long periods of time. After long periods of asymptomatic carrier stage, a proportion of FIV-seropositive cats demonstrate clinical manifestations such as generalized lymphadenopathy, diarrhea, anorexia, fever, weight loss, chronic infection, neurologic disease, and neoplasia (Fleming et al., 1991; Hopper et al., 1989; Linenberger and Abkowitz, 1995; Pedersen et al., 1987, 1989; Shelton and Linenberger, 1995; Shelton et al., 1990, 1991; Sparkes et al., 1993). These clinical manifestations in FIV infection are similar to those in human immunode-
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ficiency virus (HIV) infection, and hematologic abnormality is one of clinical problems in both FIV and HIV infection. Peripheral blood cytopenias such as anemia, neutropenia, lymphopenia, and thrombocytopenia are frequently observed in FIV-infected cats and also in HIV-infected patients (Karcher and Frost, 1991; Katsarou et al., 2001; Marche et al., 1990; Tripathi et al., 2005a,b). However, the causes leading to such cytopenias of multiple hematopoietic lineages are not well understood in FIV-infected cats. Although the causes of these cytopenias in HIV-infected patients are clearly heterogeneous and can be attributed to the hematopoietic suppressive effects of intercurrent infections or drugs commonly used in AIDS patients, some cytopenias certainly reflect bone marrow (BM) dysfunction caused by HIV itself (Bahner et al., 1997; Freedman et al., 1991; Karcher and Frost, 1991; Kojouharoff et al., 1991; Marche et al., 1990; Sun et al., 1989; Tripathi et al., 2005b; Zucker-Franklin and Cao, 1989). BM biopsy findings of HIV-infected patients are often nonspecifically abnormal. The mechanisms leading to cytopenia of diverse hematopoietic lineages caused by HIV are still unclear, however, several potential mechanisms have been proposed including the effect of viral protein such as that HIVVpr can initiate activation of BM phagocytes resulting in phagocytosis of hematopoietic cells (Kulkosky et al., 2000), immune-mediated destruction of blood cells, and direct HIV infection of hematopoietic precursors. Nowadays, it is thought that the greatest impact of HIV infection on growth and differentiation of hematopoietic progenitors results from the capacity of the virus to perturb the hematopoietic regulatory function of BM stromal cells, which are essential for normal hematopoiesis (Kulkosky et al., 2000; Moses et al., 1998). FIV infection in cats causes a loss of normal cellular composition of the bone marrow (Linenberger and Abkowitz, 1995; Shelton and Linenberger, 1995; Shelton et al., 1990, 1991, 1995). In some studies, the appearance of virus-infected BM stromal cells was suggested to be associated with severe neutropenia in acutely FIV-infected cats (Beebe et al., 1992; Linenberger et al., 1995), and recently it was reported that FIV-infection of BM stromal cells altered normal hematopoietic function (Tanabe and Yamamoto, 2001). In the present study, we investigated the prevalence of peripheral blood cytopenia observed in FIV-infected asymptomatic cats and characterized the BM cytopathology. Moreover, detection of FIV-infected cells in the BM specimens and cultured BM stromal cells was carried out to investigate the pathogenesis of peripheral blood cytopenias in FIV infection. 2. Materials and methods 2.1. Evaluation of hemograms in FIV-infected cats FIV-seropositive cats referred to the Veterinary Medical Center of the University of Tokyo were examined for complete blood count. All cats were tested for the presence of both anti-FIV antibodies and feline leukemia virus (FeLV) antigens in plasma by enzyme-linked immunosorbent-based assay (SNAP FIV/FeLV Combo Test Kit, IDEXX
Corp, Portland, ME). FIV-infected cases with FeLV infection or any other cause of peripheral blood cytopenia than FIV infection were excluded. Especially, serological analyses for other feline viruses, PCR for hemotropic mycoplasmas, and imaging radiography and ultrasonography were performed routinely. Subsequently, 50 cats showing no clinical symptom related to immunodeficiency, such as concurrent infection and inflammatory and neoplastic lesions, were included in this study. The smallest cell count of each blood cell lineage during the period of admission was recorded. Peripheral blood smears stained with Wright-Giemsa solution were evaluated for the cytology and leukograms. If anemia was observed, peripheral blood smears of the cat were stained with new methylene blue solution to evaluate reticulocyte production. Anemia was defined as a hematocrit less than 30%, leukopenia was defined as a leukocyte count less than 5.5 109/L, neutropenia was defined as a neutrophil count less than 3.0 109/L, lymphopenia was defined as a lymphocyte count less than 1.0 109/L, and thrombocytopenia was defined as a platelet count less than 150 109/L. If the cat was anemic, nonregenerative anemia was defined as an aggregate and a punctuate reticulocyte counts less than 42 109/L and 200 109/L, respectively. Prevalence of each cytopenia was investigated. 2.2. BM evaluation BM aspirate smears and biopsies were evaluated in 8 of the 50 FIV-infected cats in which cytopenias were not conceivably caused by any obvious cause other than FIV infection. BM aspirate and biopsy samples were taken from the proximal femurs or the proximal humeruses with the use of BM biopsy needle. BM aspirate smears stained with Wright-Giemsa stain were evaluated for the cytology. BM biopsy samples were fixed in buffered 10% formalin and subjected to histopathology to evaluate cellularity by staining with hematoxylin-eosin stain. BM evaluation of 3 normal cats was also performed to make comparison. BM aspiration samples taken from the 8 FIV-infected cats with peripheral blood cytopenias were subjected to detection of FIV-infected cells by immunocytochemistry (ICC) and polymerase chain reaction (PCR). 2.3. Cultivation of stromal monolayers from BM cultures Parts of BM aspirated cells were used for preparation of BM cultures. The BM culture system performed in this study is a modified method of standard procedure in humans and cats (Bahner et al., 1997; Linenberger et al., 1995; Schwartz et al., 1994, 1995; Tanabe and Yamamoto, 2001). Aspirated BM cells were washed three times in phosphate buffered saline (PBS), and resuspended in BM culture medium consisting of Dulbecco’s modified eagle’s medium (DMEM), 15% fetal bovine serum, 15% horse serum, 2-mercaptoethanol (10 4 mol/L), hydrocortisone (10 6 mol/L), and 0.05 mg of penicillin and 0.05 mg of streptomycin per mL. After cultivation for 4–5 days, the adherent cells were extensively washed with PBS to remove nonadherent cells and cultured in fresh BM culture medium until the cell monolayer was confluent. Stromal
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cells cultured for 10 days after the BM aspiration were used for detection of FIV-infected cells by ICC and PCR. Stromal cells grown on chamber slides were subjected to ICC. 2.4. Cytochemical and immunocytochemical analyses BM aspirate smears and cultured BM stromal cells were used for cytochemical and immunocytochemical analyses. These sample slides were fixed with buffered formalinacetone fixative (8.75% formaldehyde) for 3 min at 20 8C. The slides were washed with PBS and then subjected to cytochemistry and ICC with labeled streptavidin-biotinylated peroxidase reaction. Cytochemistry for myeloperoxidase, a-naphthyl butyrate esterase (aNBE) and naphthol AS-D chloracetate esterase (NASDCE) was performed to determine the cell lineage based on the methods described previously (Fujino et al., 2004). To detect FIV-infected cells, mouse monoclonal antibodies for FIV p24 (Biogenesis Inc., Kingstone, NH) were used as FIV-specific primary antibodies for ICC. A lymphoid cell line, FL-4 (Yamamoto et al., 1991), chronically infected with FIV Petaluma strain was used as a positive control. BM smears obtained from 3 normal cats were used as negative controls. Carazzi’s hematoxylin was used for counterstain. 2.5. Detection of integrated FIV provirus DNA by PCR DNA was extracted using QIAamp DNA Mini Kit (QIAGEN Inc., Valencia, CA) from BM aspirated cells, cultured BM stromal cells, and FL-4 cells as a positive control for FIV infection. PCR amplification was performed in a reaction mixture containing 10 mM Tris–HCl (pH8.3), 50 mM KCl, 1.7 mM MgCl2, 1% dimethyl sulfoxide, 0.2 mM each deoxynucleoside triphosphates, 1.5 units of Taq polymerase, 0.4 mM each primer and 1 mg of template DNA. The PCR primers for FIV provirus DNA and an internal control were prepared as described previously (Endo et al., 2000). PCR amplification was carried out by 30 cycles of denaturation (1 min at 94 8C), annealing (1 min at 55 8C) and polymerization (1 min at 72 8C). The resulting products were analyzed by 2% agarose gel electrophoresis. 3. Results 3.1. Prevalence of peripheral blood cytopenia in FIV-infected asymptomatic cats Fifty FIV-seropositive asymptomatic cats were examined: 32 males in which 24 cats were castrated, and 18 females in which 10 cats were spayed. The ages of the cats ranged from 11 months to 16 years. Forty-five cats were mongrel, 3 cats were American short hair, 1 cat was Persian, and 1 cat was Himalayan. Hematocrits, leukocyte counts and platelet counts ranged from 8 to 48% (median, 24%), 0.4 to 50.2 109/L (median, 6.9 109/L) and 0 to 915 109/L (median, 120 109/L), respectively. Anemia, leukopenia and thrombocytopenia were observed in 15 of 50 cats (30%), 13 of 50 cats (26%) and 12 of 50 cats (24%), respectively. All cats with anemia and leukopenia showed nonregenerative (normochromic and normocytic) anemia and neutropenia, respectively. Some of
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erythrocytes in the all anemic cats were morphologically abnormal, such as normochromic macroerythrocytes (not reticulocytes) or poikilocytes. Of the 13 cats with neutropenia, 8 have left-shift neutrophils (banded neutrophils more than 300 106/L) and other 5 have hypersegmented neutrophils predominantly. Lymphopenia was observed in 6 of the 13 cats with leukopenia. In cats with thrombocytopenia, megathrombocytosis and hypogranulated platelets were observed. Prevalence of cytopenia of only one blood lineage were ranged from 6 to 10% (only anemia or neutropenia, 10% each; only thrombocytopenia, 6%), those of bicytopenia were 4 to 6% (only anemia and neutropenia, 6%; only anemia or neutropenia and thrombocytopenia, 4% each), and that of pancytopenia was 8%. Twenty-four of fifty cats presented cytopenia and the overall prevalence of any cytopenia in FIV-infected cats was 48%. 3.2. Bone marrow evaluation of 8 FIV-infected cats Eight FIV-infected cats with no evident causes of cytopenias except FIV infection could be examined for the BM aspiration biopsy for the assessment of the hematological abnormalities. Clinical and hematological status of the cats was shown in Table 1. Ages of these 8 cats ranged from 11 months to 13 years. The younger 2 cats were infected with FIV since their births recorded on medical history. Peripheral blood cell counts of each lineage listed in Table 1 were measured on the day when BM aspirations were performed. Four of them (Case 1, 2, 4 and 8) had pancytopenia, one (Case 3) had anemia and thrombocytopenia, one (Case 5) had mild anemia and leukopenia, one (Case 6) had leukopenia only, and one (Case 7) had thrombocytopenia only. Leukopenia in 6 cats (Case 1, 2, 4, 5, 6 and 8) was generally due to neutropenia. Major microscopic abnormal findings of peripheral blood smears in these cats are showed in Fig. 1. Some of erythrocytes in the 6 anemic cats were morphologically abnormal, such as normochromic macroerythrocytes and poikilocytes (Fig. 1A and D). In the cases with severe leukopenia, severe left-shifts with the appearance of hyposegmented neutrophils or even a few myelocytes were present (Fig. 1B and C). In Case 8, hypersegmented neutrophils existed in peripheral blood despite severe neutropenia (Fig. 1A). In cases with thrombocytopenia, megathrombocytosis was observed and hypogranulated platelets were increased (Fig. 1D). The major findings of BM evaluation were summarized in Table 1 and showed in Figs. 2–4. In the BM of the 8 FIVinfected cats, myeloids/erythroids ratio ranged from 0.75 to 19, and was relatively high in Case 3 and 8 (Table 1). However, no other obvious tendency was observed in the differentiation of BM cells compared to those of normal cats (Fig. 2). BM hypocellularity was observed in one cat, normocellularity in 3 cats, and hypercellularity in 4 cats. The proportion of erythroid cells was not decreased in the FIV-infected cats examined except Case 3. In the cats, myelodysplastic changes were noted in myeloid and megakaryocytic cells whereas no marked morphological abnormality was observed in erythroid cells.
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Table 1 Clinical and hematological status of 8 FIV-infected asymptomatic cats examined for bone marrow aspiration biopsy. Case no.
Age
Sex
Breed
Ht (%)
Leu (109/L)
Plt (109/L)
1 2 3 4 5 6 7 8
9Y 1M 10Y 0M 7Y 0M 8Y 6M 4Y 1M 12Y 10M 11M 1Y 8M
SF CM SF SF CM SF F CM
Mongrel Mongrel Mongrel Mongrel Mongrel Mongrel Mongrel Mongrel
16 23 25 25 27 35 35 22
1.9 1.7 10.9 2.1 0.8 0.6 7.9 2.3
0 17 50 0 300 237 20 50
Case no.
Marrow cellurarity
M/E ratio
Erythroid cellurarity
Myeloid cellurarity
Megakaryocyte cellurarity
1 2 3 4 5 6 7 8
Hyper Hyper Hypo Normal Hyper Normal Normal Hyper
1.35 1.97 19 0.8 0.75 1.86 0.93 6.0
Hyper Hyper Hypo Normal Hyper Normal Normal Normal
*Hyper (IM) Hyper Hypo (IM) Normal *Hyper (IM) *Normal Normal *Hyper (IM)
*Hyper Hyper Hypo Hypo Hyper Hypo Normal *Hypo
CM: castrated male; F: female; SF: spayed female; Ht: hematocrit; Leu: leukocyte count; Plt: platelet count; M/E ratio: myeloids/erythroids ratio; IM: insufficient maturation observed. Asterisks indicate dysplastic changes observed.
The remarkably decreased count of segmented neutrophils compared to that of band neutrophils, considered to be a finding of insufficient maturation in cats with neutropenia, was observed in 4 (Case 1, 3, 5 and 8) of the 8 cats (Fig. 3). Of those, 3 (Case 1, 5 and 8) cats showed leukopenia (Table 1). Some features of
myeloid dysplasia were found in the 3 cats as well as Case 6, such as the appearance of giant band neutrophils (Fig. 4A and C), neutrophils with a ring-shaped nucleus (Fig. 4A), hypersegmented neutrophils instead of severe left shift in the BM (Fig. 4A), and abnormal karyorrhexis (Fig. 4B).
Fig. 1. Abnormal morphology of peripheral blood cells in FIV-infected cats. Normochromic macroerythrocytosis, poikilocytosis and a hypersegmented neutrophil are observed in Case 8 (A). A hyposegmented neutrophil with vacuoles is observed in Case 6 (B). A neutrophilic myelocyte with vacuolation is observed in Case 5 (C). Normochromic macroerythrocytosis, poikilocytosis and a hypogranulated megathrombocyte are observed in Case 1 (D). WrightGiemsa staining. Magnification 800 (A) and 1000 (B to D).
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Fig. 2. Differential myelograms of BM nucleated cells in 8 FIV-infected cats with peripheral blood cytopenia (Case 1 to 8) and 3 normal cats without FIVinfection (Case 9 to 11). Clinical and hematological status and BM findings are listed in Table 2. No obvious tendencies in 8 FIV-infected cats are observed in the differentiation of BM cells compared to those in normal cats.
The number of megakaryocytic series in 3 of 6 cats with thrombocytopenia was decreased in the BM (Table 1 and Fig. 2). In Case 1 and 8 with peripheral thrombocytopenia, dysplastic changes of megakaryocytes such as increased number of involution forms, micromegakaryocytes (Fig. 4D), asynchronous maturation (Fig. 4E), and separated multinucleation (Fig. 4F) were remarkably observed. It was notable that dysplastic changes of the BM were observed in 4 (Case 1, 5, 6 and 8) of the 8 cats. Of those, 2 cats (Case 1 and 8), which showed pancytopenia, had obvious dysplasia in myeloid and megakaryocyte lineages. Hematological abnormalities of these 2 cats were consistent with the diagnostic criteria of myelodysplastic syndromes (Hisasue et al., 2001).
3.3. Detection of FIV-infected BM cells Results of detection of FIV-infected cells were shown in Table 2. PCR detection of FIV-infected cells in the primary BM aspirates was positive in all of the 8 FIV-infected cats. ICC was performed to detect FIV-infected cells in the primary BM aspirates. By ICC, 3 cats (Case 1, 2 and 8) were positive for FIV in parts of lobulated mononuclear cells (myeloids, monocytes, macrophages and lymphoids) and megakaryocytes (Fig. 5A and B). All of these 3 cats had pancytopenia. Another 2 cats (Case 5 and 6) were positive in parts of lobulated mononuclear cells. Both of these 2 cats had leukopenia. Erythroid cells positive for FIV could be observed in none of the 8 cats.
Fig. 3. Differential myelograms of BM myeloid cells in 8 FIV-infected cats with peripheral blood cytopenia (Case 1 to 8) and 3 normal cats without FIVinfection (Case 9 to 11). Eosinophilic and basophilic lineages fill very small parts of the total myeloid cells in all of the 11 cats. Asterisks indicate cats with peripheral neutropenia.
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Fig. 4. Dysplastic appearances of BM cells in FIV-infected cats. A hypersegmented neutrophil (A) appeared in spite of the decreased counts of peripheral neutrophils and segmented neutrophils in the BM in Case 8. A neutrophil with ring-shaped nucleus (A), giant band neutrophils (A), abnormal karyorrhexis of a neutrophil (B) and a dwarf megakaryocyte (D) are also observed in the cat. Giant band neutrophils (C) and megakaryocytes with asynchronous maturation (E) and separated multinuclei (F) are observed in Case 1. Wright-Giemsa staining. Magnification 800 (A), 1000 (B to D) and 200 (E and F).
Table 2 Detection of FIV-infected BM cells in 8 FIV-infected asymptomatic cats examined for bone marrow aspiration biopsy. Case no.
BM cells by PCR
BM cells by ICC
SC by PCR
SC by ICC
1 2 3 4 5 6 7 8
+ + + + + + + +
LM, MK LM, MK
+ + + + + + + +
+ + + + + + + +
LM LM LM, MK
BM: bone marrow; SC: bone marrow stromal cells; PCR: polymerase chain reaction; ICC: immunocytochemistry; +: detected; : not detected; LM: lobulated mononuclear cells (myeloids, monocytes, macrophages and lymphoids); MK: megakaryocytes.
PCR detection of FIV-infected cells in the cultured BM stromal cells was positive in all of the 8 cats. Most of the cultured BM stromal cells were large round-shaped cells which were positive for aNBE (Fig. 5C) and negative for both myeloperoxidase and NASDCE, suggested that the cells were originated from macrophages. Parts of the stromal cells were positive for FIV by ICC in all of the 8 cats (Fig. 5D). 4. Discussion In the present study, hematological status was evaluated in FIV-infected asymptomatic cats with no other causes of peripheral blood cytopenia than FIV infection. The prevalence of peripheral blood cytopenia in clinically asymptomatic cats with FIV infection was revealed
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Fig. 5. Detection of FIV-infected BM cells by ICC. Two lobulated mononuclear cells which can be recognized as band neutrophils in the BM are positive for FIV in Case 5 (A). Two megakaryocytes in the BM are positive for FIV in Case 1 (B). More than half of BM stromal cells at 10 days in culture consist of large roundshaped cells which are positive for aNBE (C) and negative for both myeloperoxidase and NASDCE, suggested that the cells are originated from macrophages. Parts of the stromal cells are positive for FIV by ICC in all of the 8 cats examined (D). Carazzi’s hematoxylin counterstaining. Magnification 1000 (A), 200 (B) and 400 (C and D).
initiatively in this study. In previous studies, 71–75% of FIV-infected cats including symptomatic patients related to immunodeficiency showed hematological abnormality in some lineage (Fleming et al., 1991; Hopper et al., 1989; Shelton et al., 1990; Sparkes et al., 1993). Anemia, neutropenia and thrombocytopenia were observed in 18 to 36%, 11 to 34% and 8% in the studies, respectively. Cytopenia in multiple lineage was reported commonly in the cats. In the previous and present studies, it can be suggested that cytopenia is a common finding in FIVinfected cats not only clinically symptomatic but also asymptomatic, and cytopenia of more than one lineage is more common in cytopenic FIV-infected cats. Cytopenia in the present cases might be directly or indirectly caused by virus infection since the cats showed no other cause than FIV infection. Likewise, HIV-infected human patients, who had no other cause than HIV infection, incidentally suffered from peripheral cytopenia even if they were asymptomatic (Coyle, 1997; Kulkosky et al., 2000; Saba et al., 1995; Shelton et al., 1990; Tripathi et al., 2005a). Cytopathological findings of BM biopsies from the FIV-infected cats with peripheral blood cytopenia showed similar abnormalities to those from HIV-infected human patients. HIV-related BM abnormalities can be observed as dysplasia of erythroid, myeloid and/or megakaryocytic lineages and similar to those of myelodysplastic syndromes (MDS). The BM lesions accompanying HIV have been considered to be distinguished from
MDS in pathogenesis and diagnosed as HIV-myelopathy (Katsarou et al., 2001; Tripathi et al., 2005a,b). FIVrelated BM abnormalities have been observed as megaloblastic erythropoiesis (Shelton et al., 1990, 1995) and granulocytic dysplasia (Beebe et al., 1992) previously, as well as dysplasia of myeloid and megakaryocytic lineages in this study. In FIV-infected cats with hematological manifestations, myeloid immaturity and myelodysplastic changes have been observed in parts of the cats in the present and previous (Beebe et al., 1992; Shelton et al., 1990) studies and the changes can be consistent with the diagnostic features of feline MDS (Hisasue et al., 2001). FeLV-related MDS in cats have been considered as clonal malignancies (Hisasue et al., 2000) and sometimes developed into acute myeloid leukemia (AML) as shown in human MDS. Since none of the FIV-infected cats with myelodysplasia developed AML in this study, FIV-related myelodysplasia might be different from FeLV-related MDS in pathogenesis and be diagnosed as FIV-myelopathy. Analysis for genetic clonality of the myelodysplasia in the cats can be required to determine the theme. To determine FIV-infected cells, ICC and PCR for detection of FIV in BM cells and cultured BM stromal cells were performed in the present 8 cats. Integrated FIV proviruses in cellular genomes were detected by PCR in BM cells of all the cats. By ICC, parts of lobulated mononuclear cells and megakaryocytes were positive for FIV in cats with leukopenia and thrombocytopenia, respectively, whereas
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none of erythroid cells was detected for FIV in the cats. The present findings of FIV-infected cells in BM by ICC were consistent with the previous study to detect FIV RNA by in situ hybridization (Beebe et al., 1992) and resembled to those in HIV (Freedman et al., 1991; Kojouharoff et al., 1991; Sun et al., 1989; Zucker-Franklin and Cao, 1989). Meanwhile, parts of cultured BM stromal cells in all of the 8 cats were also positive for FIV by both PCR and ICC in this study. BM stromal cells are infectious targets for FIV and the infection may have potential to cause BM failure (Linenberger et al., 1995; Linenberger and Deng, 1999; Tanabe and Yamamoto, 2001), likewise in HIV infection (Bahner et al., 1997; Kulkosky et al., 2000; Moses et al., 1998; Schwartz et al., 1994, 1995). Reviewing the present and previous studies, multifactorial mechanisms of peripheral blood cytopenia in FIV infection may be proposed as follows: (1) FIV infection into hematopoietic precursors and BM stromal cells may induce myelodysplastic changes to cause ineffective hematopoiesis (Beebe et al., 1992; Shelton et al., 1990). (2) FIV-infected BM stromal cells can alter hematopoietic ability by growth inhibition of BM cells (Linenberger et al., 1995; Linenberger and Deng, 1999; Tanabe and Yamamoto, 2001). (3) FIV-related immune modulation may cause immune-mediated destruction of both infected and uninfected blood cells, like as observed in HIV infection (Coyle, 1997; Kulkosky et al., 2000; Saba et al., 1995). In conclusion, the hematological manifestations in FIV-infected asymptomatic cats are similar to those in HIV-infected asymptomatic patients. The present observation can be useful for understanding clinical symptoms in FIV-infected cats and for further studies on hematological abnormalities in FIV infection, as well as in HIV infection as an animal model. Acknowledgements The authors were grateful to Mrs. Akane Fujino for helpful discussions. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan through a Grant-in Aid for Scientific Research. References Bahner, I., Kearns, K., Coutinho, S., Leonard, E.H., Kohn, D.B., 1997. Infection of human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-induced hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma. Blood 90, 1787–1798. Beebe, A.M., Gluckstern, T.G., George, J., Pedersen, N.C., Dandekar, S., 1992. Detection of feline immunodeficiency virus infection in bone marrow of cats. Vet. Immunol. Immunopathol. 35, 37–49. Coyle, T.E., 1997. Hematologic complications of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. Med. Clin. North Am. 81, 449–470. Endo, Y., Goto, Y., Nishimura, Y., Mizuno, T., Watari, T., Hasegawa, A., Hohdatsu, T., Koyama, H., Tsujimoto, H., 2000. Inhibitory effect of stromal cell derived factor-1 on the replication of divergent strains of feline immunodeficiency virus in a feline T-lymphoid cell line. Vet. Immunol. Immunopathol. 74, 303–314. Fleming, E.J., McCaw, D.L., Smith, J.A., Buening, G.M., Johnson, C., 1991. Clinical, hematologic, and survival data from cats infected with feline immunodeficiency virus: 42 cases (1983–1988). J. Am. Vet. Med. Assoc. 199, 913–916.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Prevalence of hematological abnormalities and detection of infected bone marrow cells in asymptomatic cats with feline immunodeficiency virus infection Yasuhito Fujino 1,*, Hiroshi Horiuchi 1, Fuminori Mizukoshi, Kenji Baba, Yuko Goto-Koshino, Koichi Ohno, Hajime Tsujimoto Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, the University of Tokyo, Tokyo 113-8657, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 June 2008 Received in revised form 31 October 2008 Accepted 4 November 2008
Peripheral blood cytopenia such as anemia, leukopenia with neutropenia and thrombocytopenia is frequently observed in cats infected with feline immunodeficiency virus (FIV). Although previous studies report that cytopenia has been observed in FIVinfected symptomatic cats, yet the asymptomatic cats also present cytopenia occasionally. In the present study, hematological and virological analyses in FIV-infected asymptomatic cats were carried out to understand the prevalence and pathogenesis of peripheral blood cytopenia in FIV infection. Hematological abnormalities were detected in 24 of 50 FIVinfected asymptomatic cats (48%) in which no other cause of cytopenia than FIV infection was observed. Anemia only, neutropenia only, thrombocytopenia only, bicytopenia and pancytopenia were observed in 10%, 10%, 6%, 14% and 8%, respectively. Bone marrow (BM) examination was performed in 8 FIV-infected asymptomatic cats with peripheral blood cytopenia. Myeloid dysplasia was observed in 4 cats with neutropenia of which 2 cats with concurrent thrombocytopenia presented morphological abnormalities of megakaryocytes. FIV-infected BM cells in the 8 cats were analyzed by PCR and immunocytochemistry. Lobulated mononuclear cells in BM were infected with FIV in 5 cats with neutropenia of which 2 cats with concurrent thrombocytopenia showed FIV-infected megakaryocytes. Parts of isolated stromal cells from BM were infected with FIV in all the 8 cats. Present results suggest that FIV infection of BM cells can cause peripheral blood cytopenia and myelodysplasia even if the cat is asymptomatic. Such FIV-related hematological abnormalities are supposed to be diagnosed as FIV-myelopathy. ß 2008 Published by Elsevier B.V.
Keywords: FIV Cat Peripheral blood cytopenia Myelodysplasia Stromal cells
1. Introduction Feline immunodeficiency virus (FIV) is a retrovirus, subfamily Lentivirinae, associated with acquired immu-
* Corresponding author at: Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: +81 3 5841 8004; fax: +81 3 5841 8178. E-mail address: [email protected] (Y. Fujino). 1 These authors contributed equally to this work. 0378-1135/$ – see front matter ß 2008 Published by Elsevier B.V. doi:10.1016/j.vetmic.2008.11.007
nodeficiency syndrome (AIDS) in cats (Pedersen et al., 1987, 1989). Cats infected with FIV may remain asymptomatic for long periods of time. After long periods of asymptomatic carrier stage, a proportion of FIV-seropositive cats demonstrate clinical manifestations such as generalized lymphadenopathy, diarrhea, anorexia, fever, weight loss, chronic infection, neurologic disease, and neoplasia (Fleming et al., 1991; Hopper et al., 1989; Linenberger and Abkowitz, 1995; Pedersen et al., 1987, 1989; Shelton and Linenberger, 1995; Shelton et al., 1990, 1991; Sparkes et al., 1993). These clinical manifestations in FIV infection are similar to those in human immunode-
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ficiency virus (HIV) infection, and hematologic abnormality is one of clinical problems in both FIV and HIV infection. Peripheral blood cytopenias such as anemia, neutropenia, lymphopenia, and thrombocytopenia are frequently observed in FIV-infected cats and also in HIV-infected patients (Karcher and Frost, 1991; Katsarou et al., 2001; Marche et al., 1990; Tripathi et al., 2005a,b). However, the causes leading to such cytopenias of multiple hematopoietic lineages are not well understood in FIV-infected cats. Although the causes of these cytopenias in HIV-infected patients are clearly heterogeneous and can be attributed to the hematopoietic suppressive effects of intercurrent infections or drugs commonly used in AIDS patients, some cytopenias certainly reflect bone marrow (BM) dysfunction caused by HIV itself (Bahner et al., 1997; Freedman et al., 1991; Karcher and Frost, 1991; Kojouharoff et al., 1991; Marche et al., 1990; Sun et al., 1989; Tripathi et al., 2005b; Zucker-Franklin and Cao, 1989). BM biopsy findings of HIV-infected patients are often nonspecifically abnormal. The mechanisms leading to cytopenia of diverse hematopoietic lineages caused by HIV are still unclear, however, several potential mechanisms have been proposed including the effect of viral protein such as that HIVVpr can initiate activation of BM phagocytes resulting in phagocytosis of hematopoietic cells (Kulkosky et al., 2000), immune-mediated destruction of blood cells, and direct HIV infection of hematopoietic precursors. Nowadays, it is thought that the greatest impact of HIV infection on growth and differentiation of hematopoietic progenitors results from the capacity of the virus to perturb the hematopoietic regulatory function of BM stromal cells, which are essential for normal hematopoiesis (Kulkosky et al., 2000; Moses et al., 1998). FIV infection in cats causes a loss of normal cellular composition of the bone marrow (Linenberger and Abkowitz, 1995; Shelton and Linenberger, 1995; Shelton et al., 1990, 1991, 1995). In some studies, the appearance of virus-infected BM stromal cells was suggested to be associated with severe neutropenia in acutely FIV-infected cats (Beebe et al., 1992; Linenberger et al., 1995), and recently it was reported that FIV-infection of BM stromal cells altered normal hematopoietic function (Tanabe and Yamamoto, 2001). In the present study, we investigated the prevalence of peripheral blood cytopenia observed in FIV-infected asymptomatic cats and characterized the BM cytopathology. Moreover, detection of FIV-infected cells in the BM specimens and cultured BM stromal cells was carried out to investigate the pathogenesis of peripheral blood cytopenias in FIV infection. 2. Materials and methods 2.1. Evaluation of hemograms in FIV-infected cats FIV-seropositive cats referred to the Veterinary Medical Center of the University of Tokyo were examined for complete blood count. All cats were tested for the presence of both anti-FIV antibodies and feline leukemia virus (FeLV) antigens in plasma by enzyme-linked immunosorbent-based assay (SNAP FIV/FeLV Combo Test Kit, IDEXX
Corp, Portland, ME). FIV-infected cases with FeLV infection or any other cause of peripheral blood cytopenia than FIV infection were excluded. Especially, serological analyses for other feline viruses, PCR for hemotropic mycoplasmas, and imaging radiography and ultrasonography were performed routinely. Subsequently, 50 cats showing no clinical symptom related to immunodeficiency, such as concurrent infection and inflammatory and neoplastic lesions, were included in this study. The smallest cell count of each blood cell lineage during the period of admission was recorded. Peripheral blood smears stained with Wright-Giemsa solution were evaluated for the cytology and leukograms. If anemia was observed, peripheral blood smears of the cat were stained with new methylene blue solution to evaluate reticulocyte production. Anemia was defined as a hematocrit less than 30%, leukopenia was defined as a leukocyte count less than 5.5 109/L, neutropenia was defined as a neutrophil count less than 3.0 109/L, lymphopenia was defined as a lymphocyte count less than 1.0 109/L, and thrombocytopenia was defined as a platelet count less than 150 109/L. If the cat was anemic, nonregenerative anemia was defined as an aggregate and a punctuate reticulocyte counts less than 42 109/L and 200 109/L, respectively. Prevalence of each cytopenia was investigated. 2.2. BM evaluation BM aspirate smears and biopsies were evaluated in 8 of the 50 FIV-infected cats in which cytopenias were not conceivably caused by any obvious cause other than FIV infection. BM aspirate and biopsy samples were taken from the proximal femurs or the proximal humeruses with the use of BM biopsy needle. BM aspirate smears stained with Wright-Giemsa stain were evaluated for the cytology. BM biopsy samples were fixed in buffered 10% formalin and subjected to histopathology to evaluate cellularity by staining with hematoxylin-eosin stain. BM evaluation of 3 normal cats was also performed to make comparison. BM aspiration samples taken from the 8 FIV-infected cats with peripheral blood cytopenias were subjected to detection of FIV-infected cells by immunocytochemistry (ICC) and polymerase chain reaction (PCR). 2.3. Cultivation of stromal monolayers from BM cultures Parts of BM aspirated cells were used for preparation of BM cultures. The BM culture system performed in this study is a modified method of standard procedure in humans and cats (Bahner et al., 1997; Linenberger et al., 1995; Schwartz et al., 1994, 1995; Tanabe and Yamamoto, 2001). Aspirated BM cells were washed three times in phosphate buffered saline (PBS), and resuspended in BM culture medium consisting of Dulbecco’s modified eagle’s medium (DMEM), 15% fetal bovine serum, 15% horse serum, 2-mercaptoethanol (10 4 mol/L), hydrocortisone (10 6 mol/L), and 0.05 mg of penicillin and 0.05 mg of streptomycin per mL. After cultivation for 4–5 days, the adherent cells were extensively washed with PBS to remove nonadherent cells and cultured in fresh BM culture medium until the cell monolayer was confluent. Stromal
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cells cultured for 10 days after the BM aspiration were used for detection of FIV-infected cells by ICC and PCR. Stromal cells grown on chamber slides were subjected to ICC. 2.4. Cytochemical and immunocytochemical analyses BM aspirate smears and cultured BM stromal cells were used for cytochemical and immunocytochemical analyses. These sample slides were fixed with buffered formalinacetone fixative (8.75% formaldehyde) for 3 min at 20 8C. The slides were washed with PBS and then subjected to cytochemistry and ICC with labeled streptavidin-biotinylated peroxidase reaction. Cytochemistry for myeloperoxidase, a-naphthyl butyrate esterase (aNBE) and naphthol AS-D chloracetate esterase (NASDCE) was performed to determine the cell lineage based on the methods described previously (Fujino et al., 2004). To detect FIV-infected cells, mouse monoclonal antibodies for FIV p24 (Biogenesis Inc., Kingstone, NH) were used as FIV-specific primary antibodies for ICC. A lymphoid cell line, FL-4 (Yamamoto et al., 1991), chronically infected with FIV Petaluma strain was used as a positive control. BM smears obtained from 3 normal cats were used as negative controls. Carazzi’s hematoxylin was used for counterstain. 2.5. Detection of integrated FIV provirus DNA by PCR DNA was extracted using QIAamp DNA Mini Kit (QIAGEN Inc., Valencia, CA) from BM aspirated cells, cultured BM stromal cells, and FL-4 cells as a positive control for FIV infection. PCR amplification was performed in a reaction mixture containing 10 mM Tris–HCl (pH8.3), 50 mM KCl, 1.7 mM MgCl2, 1% dimethyl sulfoxide, 0.2 mM each deoxynucleoside triphosphates, 1.5 units of Taq polymerase, 0.4 mM each primer and 1 mg of template DNA. The PCR primers for FIV provirus DNA and an internal control were prepared as described previously (Endo et al., 2000). PCR amplification was carried out by 30 cycles of denaturation (1 min at 94 8C), annealing (1 min at 55 8C) and polymerization (1 min at 72 8C). The resulting products were analyzed by 2% agarose gel electrophoresis. 3. Results 3.1. Prevalence of peripheral blood cytopenia in FIV-infected asymptomatic cats Fifty FIV-seropositive asymptomatic cats were examined: 32 males in which 24 cats were castrated, and 18 females in which 10 cats were spayed. The ages of the cats ranged from 11 months to 16 years. Forty-five cats were mongrel, 3 cats were American short hair, 1 cat was Persian, and 1 cat was Himalayan. Hematocrits, leukocyte counts and platelet counts ranged from 8 to 48% (median, 24%), 0.4 to 50.2 109/L (median, 6.9 109/L) and 0 to 915 109/L (median, 120 109/L), respectively. Anemia, leukopenia and thrombocytopenia were observed in 15 of 50 cats (30%), 13 of 50 cats (26%) and 12 of 50 cats (24%), respectively. All cats with anemia and leukopenia showed nonregenerative (normochromic and normocytic) anemia and neutropenia, respectively. Some of
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erythrocytes in the all anemic cats were morphologically abnormal, such as normochromic macroerythrocytes (not reticulocytes) or poikilocytes. Of the 13 cats with neutropenia, 8 have left-shift neutrophils (banded neutrophils more than 300 106/L) and other 5 have hypersegmented neutrophils predominantly. Lymphopenia was observed in 6 of the 13 cats with leukopenia. In cats with thrombocytopenia, megathrombocytosis and hypogranulated platelets were observed. Prevalence of cytopenia of only one blood lineage were ranged from 6 to 10% (only anemia or neutropenia, 10% each; only thrombocytopenia, 6%), those of bicytopenia were 4 to 6% (only anemia and neutropenia, 6%; only anemia or neutropenia and thrombocytopenia, 4% each), and that of pancytopenia was 8%. Twenty-four of fifty cats presented cytopenia and the overall prevalence of any cytopenia in FIV-infected cats was 48%. 3.2. Bone marrow evaluation of 8 FIV-infected cats Eight FIV-infected cats with no evident causes of cytopenias except FIV infection could be examined for the BM aspiration biopsy for the assessment of the hematological abnormalities. Clinical and hematological status of the cats was shown in Table 1. Ages of these 8 cats ranged from 11 months to 13 years. The younger 2 cats were infected with FIV since their births recorded on medical history. Peripheral blood cell counts of each lineage listed in Table 1 were measured on the day when BM aspirations were performed. Four of them (Case 1, 2, 4 and 8) had pancytopenia, one (Case 3) had anemia and thrombocytopenia, one (Case 5) had mild anemia and leukopenia, one (Case 6) had leukopenia only, and one (Case 7) had thrombocytopenia only. Leukopenia in 6 cats (Case 1, 2, 4, 5, 6 and 8) was generally due to neutropenia. Major microscopic abnormal findings of peripheral blood smears in these cats are showed in Fig. 1. Some of erythrocytes in the 6 anemic cats were morphologically abnormal, such as normochromic macroerythrocytes and poikilocytes (Fig. 1A and D). In the cases with severe leukopenia, severe left-shifts with the appearance of hyposegmented neutrophils or even a few myelocytes were present (Fig. 1B and C). In Case 8, hypersegmented neutrophils existed in peripheral blood despite severe neutropenia (Fig. 1A). In cases with thrombocytopenia, megathrombocytosis was observed and hypogranulated platelets were increased (Fig. 1D). The major findings of BM evaluation were summarized in Table 1 and showed in Figs. 2–4. In the BM of the 8 FIVinfected cats, myeloids/erythroids ratio ranged from 0.75 to 19, and was relatively high in Case 3 and 8 (Table 1). However, no other obvious tendency was observed in the differentiation of BM cells compared to those of normal cats (Fig. 2). BM hypocellularity was observed in one cat, normocellularity in 3 cats, and hypercellularity in 4 cats. The proportion of erythroid cells was not decreased in the FIV-infected cats examined except Case 3. In the cats, myelodysplastic changes were noted in myeloid and megakaryocytic cells whereas no marked morphological abnormality was observed in erythroid cells.
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Table 1 Clinical and hematological status of 8 FIV-infected asymptomatic cats examined for bone marrow aspiration biopsy. Case no.
Age
Sex
Breed
Ht (%)
Leu (109/L)
Plt (109/L)
1 2 3 4 5 6 7 8
9Y 1M 10Y 0M 7Y 0M 8Y 6M 4Y 1M 12Y 10M 11M 1Y 8M
SF CM SF SF CM SF F CM
Mongrel Mongrel Mongrel Mongrel Mongrel Mongrel Mongrel Mongrel
16 23 25 25 27 35 35 22
1.9 1.7 10.9 2.1 0.8 0.6 7.9 2.3
0 17 50 0 300 237 20 50
Case no.
Marrow cellurarity
M/E ratio
Erythroid cellurarity
Myeloid cellurarity
Megakaryocyte cellurarity
1 2 3 4 5 6 7 8
Hyper Hyper Hypo Normal Hyper Normal Normal Hyper
1.35 1.97 19 0.8 0.75 1.86 0.93 6.0
Hyper Hyper Hypo Normal Hyper Normal Normal Normal
*Hyper (IM) Hyper Hypo (IM) Normal *Hyper (IM) *Normal Normal *Hyper (IM)
*Hyper Hyper Hypo Hypo Hyper Hypo Normal *Hypo
CM: castrated male; F: female; SF: spayed female; Ht: hematocrit; Leu: leukocyte count; Plt: platelet count; M/E ratio: myeloids/erythroids ratio; IM: insufficient maturation observed. Asterisks indicate dysplastic changes observed.
The remarkably decreased count of segmented neutrophils compared to that of band neutrophils, considered to be a finding of insufficient maturation in cats with neutropenia, was observed in 4 (Case 1, 3, 5 and 8) of the 8 cats (Fig. 3). Of those, 3 (Case 1, 5 and 8) cats showed leukopenia (Table 1). Some features of
myeloid dysplasia were found in the 3 cats as well as Case 6, such as the appearance of giant band neutrophils (Fig. 4A and C), neutrophils with a ring-shaped nucleus (Fig. 4A), hypersegmented neutrophils instead of severe left shift in the BM (Fig. 4A), and abnormal karyorrhexis (Fig. 4B).
Fig. 1. Abnormal morphology of peripheral blood cells in FIV-infected cats. Normochromic macroerythrocytosis, poikilocytosis and a hypersegmented neutrophil are observed in Case 8 (A). A hyposegmented neutrophil with vacuoles is observed in Case 6 (B). A neutrophilic myelocyte with vacuolation is observed in Case 5 (C). Normochromic macroerythrocytosis, poikilocytosis and a hypogranulated megathrombocyte are observed in Case 1 (D). WrightGiemsa staining. Magnification 800 (A) and 1000 (B to D).
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Fig. 2. Differential myelograms of BM nucleated cells in 8 FIV-infected cats with peripheral blood cytopenia (Case 1 to 8) and 3 normal cats without FIVinfection (Case 9 to 11). Clinical and hematological status and BM findings are listed in Table 2. No obvious tendencies in 8 FIV-infected cats are observed in the differentiation of BM cells compared to those in normal cats.
The number of megakaryocytic series in 3 of 6 cats with thrombocytopenia was decreased in the BM (Table 1 and Fig. 2). In Case 1 and 8 with peripheral thrombocytopenia, dysplastic changes of megakaryocytes such as increased number of involution forms, micromegakaryocytes (Fig. 4D), asynchronous maturation (Fig. 4E), and separated multinucleation (Fig. 4F) were remarkably observed. It was notable that dysplastic changes of the BM were observed in 4 (Case 1, 5, 6 and 8) of the 8 cats. Of those, 2 cats (Case 1 and 8), which showed pancytopenia, had obvious dysplasia in myeloid and megakaryocyte lineages. Hematological abnormalities of these 2 cats were consistent with the diagnostic criteria of myelodysplastic syndromes (Hisasue et al., 2001).
3.3. Detection of FIV-infected BM cells Results of detection of FIV-infected cells were shown in Table 2. PCR detection of FIV-infected cells in the primary BM aspirates was positive in all of the 8 FIV-infected cats. ICC was performed to detect FIV-infected cells in the primary BM aspirates. By ICC, 3 cats (Case 1, 2 and 8) were positive for FIV in parts of lobulated mononuclear cells (myeloids, monocytes, macrophages and lymphoids) and megakaryocytes (Fig. 5A and B). All of these 3 cats had pancytopenia. Another 2 cats (Case 5 and 6) were positive in parts of lobulated mononuclear cells. Both of these 2 cats had leukopenia. Erythroid cells positive for FIV could be observed in none of the 8 cats.
Fig. 3. Differential myelograms of BM myeloid cells in 8 FIV-infected cats with peripheral blood cytopenia (Case 1 to 8) and 3 normal cats without FIVinfection (Case 9 to 11). Eosinophilic and basophilic lineages fill very small parts of the total myeloid cells in all of the 11 cats. Asterisks indicate cats with peripheral neutropenia.
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Fig. 4. Dysplastic appearances of BM cells in FIV-infected cats. A hypersegmented neutrophil (A) appeared in spite of the decreased counts of peripheral neutrophils and segmented neutrophils in the BM in Case 8. A neutrophil with ring-shaped nucleus (A), giant band neutrophils (A), abnormal karyorrhexis of a neutrophil (B) and a dwarf megakaryocyte (D) are also observed in the cat. Giant band neutrophils (C) and megakaryocytes with asynchronous maturation (E) and separated multinuclei (F) are observed in Case 1. Wright-Giemsa staining. Magnification 800 (A), 1000 (B to D) and 200 (E and F).
Table 2 Detection of FIV-infected BM cells in 8 FIV-infected asymptomatic cats examined for bone marrow aspiration biopsy. Case no.
BM cells by PCR
BM cells by ICC
SC by PCR
SC by ICC
1 2 3 4 5 6 7 8
+ + + + + + + +
LM, MK LM, MK
+ + + + + + + +
+ + + + + + + +
LM LM LM, MK
BM: bone marrow; SC: bone marrow stromal cells; PCR: polymerase chain reaction; ICC: immunocytochemistry; +: detected; : not detected; LM: lobulated mononuclear cells (myeloids, monocytes, macrophages and lymphoids); MK: megakaryocytes.
PCR detection of FIV-infected cells in the cultured BM stromal cells was positive in all of the 8 cats. Most of the cultured BM stromal cells were large round-shaped cells which were positive for aNBE (Fig. 5C) and negative for both myeloperoxidase and NASDCE, suggested that the cells were originated from macrophages. Parts of the stromal cells were positive for FIV by ICC in all of the 8 cats (Fig. 5D). 4. Discussion In the present study, hematological status was evaluated in FIV-infected asymptomatic cats with no other causes of peripheral blood cytopenia than FIV infection. The prevalence of peripheral blood cytopenia in clinically asymptomatic cats with FIV infection was revealed
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Fig. 5. Detection of FIV-infected BM cells by ICC. Two lobulated mononuclear cells which can be recognized as band neutrophils in the BM are positive for FIV in Case 5 (A). Two megakaryocytes in the BM are positive for FIV in Case 1 (B). More than half of BM stromal cells at 10 days in culture consist of large roundshaped cells which are positive for aNBE (C) and negative for both myeloperoxidase and NASDCE, suggested that the cells are originated from macrophages. Parts of the stromal cells are positive for FIV by ICC in all of the 8 cats examined (D). Carazzi’s hematoxylin counterstaining. Magnification 1000 (A), 200 (B) and 400 (C and D).
initiatively in this study. In previous studies, 71–75% of FIV-infected cats including symptomatic patients related to immunodeficiency showed hematological abnormality in some lineage (Fleming et al., 1991; Hopper et al., 1989; Shelton et al., 1990; Sparkes et al., 1993). Anemia, neutropenia and thrombocytopenia were observed in 18 to 36%, 11 to 34% and 8% in the studies, respectively. Cytopenia in multiple lineage was reported commonly in the cats. In the previous and present studies, it can be suggested that cytopenia is a common finding in FIVinfected cats not only clinically symptomatic but also asymptomatic, and cytopenia of more than one lineage is more common in cytopenic FIV-infected cats. Cytopenia in the present cases might be directly or indirectly caused by virus infection since the cats showed no other cause than FIV infection. Likewise, HIV-infected human patients, who had no other cause than HIV infection, incidentally suffered from peripheral cytopenia even if they were asymptomatic (Coyle, 1997; Kulkosky et al., 2000; Saba et al., 1995; Shelton et al., 1990; Tripathi et al., 2005a). Cytopathological findings of BM biopsies from the FIV-infected cats with peripheral blood cytopenia showed similar abnormalities to those from HIV-infected human patients. HIV-related BM abnormalities can be observed as dysplasia of erythroid, myeloid and/or megakaryocytic lineages and similar to those of myelodysplastic syndromes (MDS). The BM lesions accompanying HIV have been considered to be distinguished from
MDS in pathogenesis and diagnosed as HIV-myelopathy (Katsarou et al., 2001; Tripathi et al., 2005a,b). FIVrelated BM abnormalities have been observed as megaloblastic erythropoiesis (Shelton et al., 1990, 1995) and granulocytic dysplasia (Beebe et al., 1992) previously, as well as dysplasia of myeloid and megakaryocytic lineages in this study. In FIV-infected cats with hematological manifestations, myeloid immaturity and myelodysplastic changes have been observed in parts of the cats in the present and previous (Beebe et al., 1992; Shelton et al., 1990) studies and the changes can be consistent with the diagnostic features of feline MDS (Hisasue et al., 2001). FeLV-related MDS in cats have been considered as clonal malignancies (Hisasue et al., 2000) and sometimes developed into acute myeloid leukemia (AML) as shown in human MDS. Since none of the FIV-infected cats with myelodysplasia developed AML in this study, FIV-related myelodysplasia might be different from FeLV-related MDS in pathogenesis and be diagnosed as FIV-myelopathy. Analysis for genetic clonality of the myelodysplasia in the cats can be required to determine the theme. To determine FIV-infected cells, ICC and PCR for detection of FIV in BM cells and cultured BM stromal cells were performed in the present 8 cats. Integrated FIV proviruses in cellular genomes were detected by PCR in BM cells of all the cats. By ICC, parts of lobulated mononuclear cells and megakaryocytes were positive for FIV in cats with leukopenia and thrombocytopenia, respectively, whereas
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none of erythroid cells was detected for FIV in the cats. The present findings of FIV-infected cells in BM by ICC were consistent with the previous study to detect FIV RNA by in situ hybridization (Beebe et al., 1992) and resembled to those in HIV (Freedman et al., 1991; Kojouharoff et al., 1991; Sun et al., 1989; Zucker-Franklin and Cao, 1989). Meanwhile, parts of cultured BM stromal cells in all of the 8 cats were also positive for FIV by both PCR and ICC in this study. BM stromal cells are infectious targets for FIV and the infection may have potential to cause BM failure (Linenberger et al., 1995; Linenberger and Deng, 1999; Tanabe and Yamamoto, 2001), likewise in HIV infection (Bahner et al., 1997; Kulkosky et al., 2000; Moses et al., 1998; Schwartz et al., 1994, 1995). Reviewing the present and previous studies, multifactorial mechanisms of peripheral blood cytopenia in FIV infection may be proposed as follows: (1) FIV infection into hematopoietic precursors and BM stromal cells may induce myelodysplastic changes to cause ineffective hematopoiesis (Beebe et al., 1992; Shelton et al., 1990). (2) FIV-infected BM stromal cells can alter hematopoietic ability by growth inhibition of BM cells (Linenberger et al., 1995; Linenberger and Deng, 1999; Tanabe and Yamamoto, 2001). (3) FIV-related immune modulation may cause immune-mediated destruction of both infected and uninfected blood cells, like as observed in HIV infection (Coyle, 1997; Kulkosky et al., 2000; Saba et al., 1995). In conclusion, the hematological manifestations in FIV-infected asymptomatic cats are similar to those in HIV-infected asymptomatic patients. The present observation can be useful for understanding clinical symptoms in FIV-infected cats and for further studies on hematological abnormalities in FIV infection, as well as in HIV infection as an animal model. Acknowledgements The authors were grateful to Mrs. Akane Fujino for helpful discussions. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan through a Grant-in Aid for Scientific Research. References Bahner, I., Kearns, K., Coutinho, S., Leonard, E.H., Kohn, D.B., 1997. Infection of human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-induced hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma. Blood 90, 1787–1798. Beebe, A.M., Gluckstern, T.G., George, J., Pedersen, N.C., Dandekar, S., 1992. Detection of feline immunodeficiency virus infection in bone marrow of cats. Vet. Immunol. Immunopathol. 35, 37–49. Coyle, T.E., 1997. Hematologic complications of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. Med. Clin. North Am. 81, 449–470. Endo, Y., Goto, Y., Nishimura, Y., Mizuno, T., Watari, T., Hasegawa, A., Hohdatsu, T., Koyama, H., Tsujimoto, H., 2000. Inhibitory effect of stromal cell derived factor-1 on the replication of divergent strains of feline immunodeficiency virus in a feline T-lymphoid cell line. Vet. Immunol. Immunopathol. 74, 303–314. Fleming, E.J., McCaw, D.L., Smith, J.A., Buening, G.M., Johnson, C., 1991. Clinical, hematologic, and survival data from cats infected with feline immunodeficiency virus: 42 cases (1983–1988). J. Am. Vet. Med. Assoc. 199, 913–916.
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