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Keywords:

  • Anemia;
  • Neutropenia;
  • Thrombocytopenia

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Background: Nonregenerative cytopenias such as nonregenerative anemia, neutropenia, and thrombocytopenia in cats with feline leukemia virus (FeLV) antigen are assumed to be caused by the underlying FeLV infection. In addition, cats with negative FeLV antigen-test results that have cytopenias of unknown etiology often are suspected to suffer from latent FeLV infection that is responsible for the nonregenerative cytopenias.

Objective: The purpose of this study was to assess the role of latent FeLV infection by polymerase chain reaction (PCR) in bone marrow of cats with nonregenerative cytopenias that had negative FeLV antigen test results in blood.

Animals: Thirty-seven cats were included in the patient group. Inclusion criteria were (1) nonregenerative cytopenia of unknown origin and (2) negative FeLV antigen test result. Antigenemia was determined by detection of free FeLV p27 antigen by ELISA in serum. Furthermore, 7 cats with positive antigen test results with nonregenerative cytopenia were included as control group I, and 30 cats with negative antigen test results without nonregenerative cytopenia were included as control group II.

Methods: Whole blood and bone marrow samples were tested by 2 different PCR assays detecting sequences of the envelope or long terminal repeat genes. FeLV immunohistochemistry was performed in bone marrow samples.

Results: Two of the 37 cats (5.4%) in the patient group were positive on the bone marrow PCR results and thus were latently infected with FeLV.

Conclusions and Clinical Importance: The findings of this study suggest that FeLV latency is rare in cats with nonregenerative cytopenias.

Abbreviations:
DNA

deoxyribonucleic acid

env

envelope

FeLV

feline leukemia virus

FIV

feline immunodeficiency virus

IgG

immunoglobulin G

IHC

immunohistochemistry

LTR

long terminal repeat

PCR

polymerase chain reaction

spp.

species

Hematologic disorders, particularly cytopenias because of suspected myelosuppression, are a common finding in cats infected with feline leukemia virus (FeLV).1–5 Non-neoplastic hematologic disorders described in association with FeLV include anemia of myelodysplastic syndrome; aplastic anemia (pancytopenia); persistent, transient, and cyclic neutropenias; panleukopenia-like syndrome; and platelet abnormalities.5,6 According to the literature, anemia is the major non-neoplastic complication that occurs in >50% of symptomatic FeLV-infected cats.1,2,4 Reports of over 20 years ago suggest that more than two-thirds of all nonregenerative anemias in cats were the result of FeLV infection.7 They state that only 10% of FeLV-associated anemias were regenerative and caused by immune-mediated hemolysis or secondary hemotropic Mycoplasma spp. infection.8,9 The other 90% of FeLV-associated anemias were nonregenerative and caused by myelosuppression.

In cats with hematologic disorders that have negative test results on traditional FeLV antigen tests (eg, ELISA), latent FeLV infection often is suspected as being responsible for the disorder.6,10–13 However, recent studies suggest that FeLV latency, in which provirus is present in a nonreplicating form in myelomonocytic progenitor cells, is not commonly present in these conditions classically associated with FeLV.14

Polymerase chain reaction (PCR) has been used to successfully identify FeLV proviral deoxyribonucleic acid (DNA) in peripheral blood, corneal tissue, organ samples, and bone marrow15–18 and potentially allows detection of latent infection. Cats with antigen-negative and PCR-positive test results are considered to be latently infected with FeLV. Thus, in this study, PCR positive tests results are equated with latency.

The purpose of this study was to determine the prevalence and role of latent FeLV infections in cats with bone marrow disorders.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Patients

Three groups of cats derived from a total of 74 cats were investigated prospectively. Of the 74 cats, 18 were presented to the Teaching Hospital of the College of Veterinary Medicine, University of Georgia, Athens, GA, from 1998 to 2003. Fifty-six cats were presented to the Clinic of Small Animal Internal Medicine of the College of Veterinary Medicine, LMU University of Munich, Germany, from 2002 to 2008. Thirty-seven (14 from Athens, GA; 23 from Munich, Germany) cats were included in the patient group. Cats were eligible for inclusion in this group if they had nonregenerative cytopenia of unknown origin and had negative results on FeLV p27 antigen ELISA in peripheral blood. Other reasons for nonregenerative cytopenias, including drug administration, toxins, and lymphoma, were excluded by history and bone marrow cytology and histology, respectively. Cats with positive test results for feline immunodeficiency virus (FIV) antibody also were excluded. Nonregenerative cytopenia was defined as nonregenerative anemia (anemia with a PCV <30% and an absolute reticulocyte count <15,000/μL) or nonregenerative neutropenia (segmented neutrophil count <2500/μL and band neutrophil count <400/μL) or nonregenerative thrombocytopenia (platelet count <100,000/μL and no evidence of platelet consumption or destruction or blood loss), or combinations of these disorders.

Seven (1 from Athens, GA; 6 from Munich, Germany) additional cats with nonregenerative cytopenia that had positive ELISA tests results for FeLV antigen in peripheral blood were included as positive controls (control group I). Furthermore, 30 (3 from Athens, GA; 27 from Munich, Germany) cats with normal CBC results that had negative ELISA test results for FeLV antigen in peripheral blood were included negative controls (control group II).

The age of the 37 cats in the patient group ranged from 1 to 17 years, with a median age of 10 years. Eight female spayed cats, 20 male neutered cats, 1 female intact cat, and 3 male intact cats were included. Five cats were stray cats of unknown age.

All 37 cats of the patient group were presented for hematologic problems. Seventeen cats suffered from nonregenerative anemia alone (45.9%), 3 from thrombocytopenia alone (8.1%), 6 from nonregenerative anemia and nonregenerative neutropenia (16.2%), 8 from nonregenerative anemia and thrombocytopenia (21.6%), and 3 from pancytopenia (8.1%). Cats of the patient group with nonregenerative anemia had PCV ranging from 4 to 28% (median, 20%), mean corpuscular volume ranging from 36 to 65 fl (median, 43 fl), and absolute reticulocyte counts ranging from 0 to 5900/μL (median, 0/μL). Cats with nonregenerative neutropenia had neutrophil counts ranging from 0 to 470/μL (median, 380/μL) and band neutrophils ranging from 0 to 200/μL (median, 0/μL). Cats with thrombocytopenia had thrombocyte counts ranging from 20 to 98,000/μL (median, 65,000/μL).

In all cats, FeLV ELISA tests for FeLV p27 antigen were performed on serum with a commercial FIV/FeLV combination test kit.a Only cats that had positive test results, twice in separate runs, were considered to have positive FeLV antigen test results. Both tests were performed from the same blood sample, but a new test kit was used to detect a false positive result caused by an unspecific test-related reaction.

In 21 cats, bone marrow was obtained from the humerus with a 14-G bone marrow needleb under sedation. In the remaining 53 cats, bone marrow was obtained from the humerus during necropsy. Approximately 500 μL of bone marrow was placed in a Falcon tube.c Serum, blood, and bone marrow samples were immediately frozen and stored at −70°C before analysis. Laboratory specimens, including blood and bone marrow, were obtained from clinical patients at the University of Georgia, College of Veterinary Medicine Teaching Hospital and at the LMU University of Munich, Clinic of Small Animal Medicine with owner and Hospital Board approval, and by methods approved by the animal and care and use committees of these institutions.

PCR

Two different PCR assays (envelope [env] PCR and long terminal repeat [LTR] PCR) were performed to detect proviral FeLV DNA. Samples were tested either by env PCR or by LTR PCR. Because collection of samples was performed over a period of 10 years, 2 different PCR assays were utilized, because the env PCR chosen in the beginning of the study was not available over the whole study period and thus, the LTR PCR was used.

In all 74 cats, bone marrow was investigated by PCR (21 by env PCR and 53 by LTR PCR). In 56 of the 74 cats, blood was tested by PCR (20 by env PCR and 36 by LTR PCR). Blood of 33 of 37 cats of the patient group, 4 of 7 cats of the control group I, and 19 of 30 cats of the control group II was tested.

Samples were stored for a maximal period of 6 months before analysis. Isolation of genomic DNA (200 μL aliquots of blood or bone marrow) was performed with the QIAamp DNA Blood & Tissue Kit.d The env PCR used in this study was obtained from Synbiotics.e The system detected a DNA sequence of 312 bp in the env region coding for the gp70 of the FeLV genome (6110–6421 bp) that is specific for exogenous FeLV. A positive and a negative control included in the PCR kit were used.

The LTR PCR used in this study was described by Tandon et al19 and detects a sequence of the U3 region of the LTR which also is specific for exogenous FeLV. The PCR was developed in the Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Switzerland, and is now used by many laboratories.20 In every run, 1 water control and 1 positive control per 9 cat samples were included. DNA and RNA were extracted by an automated system.f The master mixes, primers and probes, and samples were pipetted by a robot.g The extractor and the robot provide excellent performance with respect to absence of cross-contamination. In the event that 1 water control was positive, the entire run was discarded.

Immunohistochemistry (IHC)

IHC of bone marrow was used to detect intracellular FeLV p27 antigen. Immunohistochemical detection of FeLV p27 antigen was performed in every cat in which an adequate sample volume of bone marrow material for PCR and IHC could be obtained. IHC was performed from 44 cats (12 of 37 cats of the patient group, 4 of 7 cats of the control group I, and 27 of 30 cats of the control group II). A polyclonal goat anti-FeLV p27 antibodyh (diluted 1 : 2000) was used as primary antibody, and a rabbit antigoat immunoglobulin G (IgG) coupled with peroxidasei (diluted 1 : 400) as a secondary antibody.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

PCR

In the control group I (positive antigen test result with nonregenerative cytopenia), all cats had positive test results for bone marrow PCR and blood PCR (Table 1). In control group II (negative antigen test result without nonregenerative cytopenia), all cats had negative results for bone marrow PCR and blood PCR. In the patient group (negative antigen test results with nonregenerative cytopenia), of the 33 cats tested by blood PCR, all had negative test results. However, 2 cats of the patient group had positive PCR results for FeLV in bone marrow and were therefore identified as latently infected. The prevalence of latent FeLV infection in this study was 5% (95% confidence interval 0.7–18.2%).

Table 1.   Results of env PCR, LTR PCR, and IHC in the 74 cats of the study.
MethodPatient Group (n=37)Control Group I (n=7)Control Group II (n=30)
  1. PCR, polymerase chain reaction; IHC, immunohistochemistry; env, envelope; LTR, long terminal repeat.

Blood env PCR
 Tested1910
 Negative1900
 Positive010
Blood LTR PCR
 Tested14319
 Negative14019
 Positive030
Bone marrow env PCR
 Tested2010
 Negative1800
 Positive210
Bone marrow LTR PCR
 Tested17630
 Negative17030
 Positive060
Bone marrow IHC
 Tested12327
 Negative12027
 Positive030

One of the 2 cats with positive results in bone marrow was a 6-year-old male neutered domestic shorthair cat that had a 1-month history of nonregenerative anemia and intermittent fever. On admission, the cat was very lethargic and febrile. Notable findings on a CBC were hypochromic, normocytic nonregenerative anemia with a PCV of 24% and a reticulocyte count of 0/μL. White blood cell and platelet counts were within reference limits. Hyperglobulinemia of 5.1 g/dL (reference range, 3.0–3.8 g/dL) was the only finding of a complete biochemical screening. Enlarged mesenteric lymph nodes were found by abdominal ultrasound examination. The bone marrow aspirate finding was severe erythroid hypoplasia suggestive of pure red cell aplasia. The cat was euthanized.

The other cat was a 7-year-old male neutered domestic shorthair cat that had chronic nonregenerative anemia. CBC findings were normochromic, normocytic nonregenerative anemia with a PCV of 15% and a reticulocyte count of 0/μL. There were 9 nucleated red blood cells per 100 white blood cells on the blood smear. The white blood cell count and platelet count were within normal limits. The bone marrow finding was erythropoietic hypoplasia. Treatment with prednisolone and metronidazole was initiated, and the cat improved clinically and was discharged from the hospital. Information from a follow-up phone call with the referring veterinarian was that the cat had a PCV of 16% 1 month later. Fourteen months later, the cat was euthanized because of severe anemia with a PCV of 9%. No FeLV test was repeated on this cat.

IHC

In control group I, 3 of 7 cats were tested. All tested cats had positive test results by IHC from bone marrow. In control group II, 27 of 30 cats were tested. All cats tested had negative test results by IHC from bone marrow. In the patient group, 12 of 37 cats were tested, and all had negative results by IHC. IHC investigation of the bone marrow of the 2 PCR-positive infected cats was not performed.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Cytopenias in the peripheral blood of cats that have FeLV-positive results for antigen by ELISA usually are attributed to the underlying FeLV infection.21,22 Various studies have demonstrated the association of anemia, leukopenia, and thrombocytopenia with FeLV infection.7,9,23–25 Cats that have FeLV antigen-negative test results with unexplained peripheral blood cytopenias often are suspected to be suffering from latent FeLV infection.5 However, Herring et al14 showed that persistent or latent infection is not always present in cats with symptoms classically associated with FeLV. They performed serum ELISA and both immunofluorescent antibody test and PCR of blood and bone marrow samples from 16 cats with diseases suspected to be FeLV-associated. Twelve cats had FeLV-negative results on all tests, 1 cat had FeLV-positive results on all tests (and thus was persistently viremic), and 3 cats had discordant test results. None of the cats with negative blood test results had positive results on bone marrow PCR indicating latent infection.14

This observation is consistent with the low prevalence of latent FeLV infection in cats with nonregenerative cytopenia detected in the present study. The prevalence in our study was 5% (95% confidence interval: 0.7–18.2%) and therefore FeLV latency was rare in cats with signs of myelosuppression. There are no comparable studies published in which FeLV latency was investigated in a large group of cats with nonregenerative cytopenias by bone marrow PCR. One recent study investigated the presence of proviral DNA in blood of unselected cats.26 In that study, 5.4% (24/445) of antigen-negative cats were positive by PCR. Considering the fact that selected cats in which FeLV infection is a major differential diagnosis were tested in the present study, a much higher rate would be expected. In addition, in the previous study, PCR was only performed on blood samples. PCR with blood is likely to be less sensitive to detect FeLV latency than PCR with bone marrow, and in the present study latency was not detected in blood samples. Therefore, prevalence in the present study seems very low. A likely reason for the low prevalence of latent FeLV infection in this study is the fact that FeLV prevalence is decreasing worldwide. Because the proportion of FeLV-positive cats in the overall population is decreasing, the proportion of FeLV-positive cats in a population of sick cats, such as the patient group of this study, is expected to decrease as well. In an older study in which a higher prevalence of FeLV antigenemia was found in anemic cats,7 the investigated cat population was younger than that of the present study. In an older population, other causes of anemia (eg, lymphoma or anemia of chronic diseases) are more likely to be present than in younger cats. In addition, the 2 PCR methods used in this study detect a highly conserved region of the FeLV provirus, but it is still possible that virus strains with mutations in this region or small remnants of the FeLV genome may have been present and remained undetected, but still caused bone marrow alteration. The genetic sequence of FeLV may differ in myelodysplastic forms of the virus although 2 sequences were tested in the present study, and sequence variation in LTR can cause different syndromes.27 The sequences for which the primers were chosen in this study could possibly not be present in potential myelodysplastic strains, or an endogenous genome from a prior retrovirus infection (ie, vertically acquired infections) might become activated by stress in the absence of exogenous type A virus to trigger myelodysplastic events via genetic promotion as suggested by Maksakova et al.28 Furthermore, the cats could have been coinfected with endogenous FeLV B or C which inhibited the expression or replication of FeLV A as described by Phipps et al.29 These strains were not specifically detected with the methods used in the present study. Recently, quantitative and nested PCR methods have been shown to increase the sensitivity of conventional PCR methods.30 Thus, the PCR as performed might have missed the latent FeLV genome in some cases. Additionally, the qualitative PCR in this study only identified exogenous viral sequences and the FeLV genome may become modified in latency and lose the exogenous genetic markers selected here. Persistent viremia has been shown to be associated with secondary viremia of bone marrow origin, whereas regressor cats only sustain a nonproductive infection in low numbers of leukocytes.31 Consequently, both viral load and cell type could have influenced the PCR results because lymphoid rather than myeloid cells might be more likely to contain latent virus.31

Although FeLV latency may be responsible for a variety of bone marrow diseases, this appears to be an uncommon event. In a study described by Pacitti,32 26 experimentally infected cats with latent FeLV infection were followed for up to 4 years. None of the 26 cats developed bone marrow-related disease. Pedersen et al33 placed 400 cats that had recovered from viremia after experimental FeLV infection in homes throughout the United States and studied their conditions in the next 6 years. Fifty-two of these cats developed a variety of minor or serious diseases, but none of them developed bone marrow disorders. Two of the cats showed FeLV viremia again (reactivation of latent infection), and 1 of these 2 cats was diagnosed with myeloproliferative disease. The proportion of experimentally infected cats that harbor latent FeLV infections in their bone marrow decreases with time after disappearance of viremia.33 The most pronounced decrease in the incidence of latent infections occurred 190 days postviremia.33,34 Three years after viremia, only approximately 8% of cats still harbor latent infection in marrow myelomonocytic cells12,33–35 and stromal fibroblast cells.36 Newer studies, however, cast doubt on the hypothesis that latent infection actually is eliminated from the body. Hofmann-Lehmann et al37 followed cats with latent infection and cats with regressive infection, and none of these cats showed permanent absence of provirus and negative PCR results turned positive in at least one of the investigated time points in all cats.

Although some studies suggest that latent FeLV infection also can be detected by PCR in blood,20 in our study blood PCR results were negative from 1 of the 2 cats of the patient group with bone marrow PCR-positive test results (the other cat's blood was not tested). This suggests the possibility that PCR of bone marrow continues to be a more sensitive diagnostic tool to detect latent FeLV infection than PCR of blood cells. This observation also may be because of the fact that blood samples contain fewer cells than do bone marrow samples per volume, which also explains the difference in sensitivity. Unfortunately, IHC investigation of the bone marrow of the 2 PCR-positive cats was not performed. In this study, all results of IHC testing corresponded with the results of the FeLV p27 antigen ELISA. Because in this study IHC of bone marrow in every case was identical with the results of the p27 antigen ELISA, IHC seems to be less sensitive than bone marrow PCR to detect latent FeLV infections. IHC was used before the invention of PCR to detect FeLV latency, but it detects antigen that only is produced when virus is replicating, which by definition is not the case during latency. This explains the lower sensitivity of IHC. In a study by Herring et al,38 IHC and PCR were performed in corneal tissue of cats with FeLV antigen-positive results. PCR showed more positive test results than IHC. The most likely explanation is the better sensitivity of PCR to detect low levels of virus compared with IHC. Alteration of gp70 antigens by formalin fixation also may decrease the sensitivity of IHC.39

Comparing all diagnostic tools used in this study, PCR from bone marrow can be considered to be the most sensitive method to detect FeLV latency.

In the present study, latent infection was detected in 2 cats and it is possible that this latent FeLV infection caused the observed nonregenerative cytopenia. However, it is also possible that FeLV infection and and cytopenia were coincidental findings because the prevalence of FeLV in this study is only slightly higher than in cats in general.40 Because bone marrow microenvironment cells provide a reservoir of latent FeLV infections in which provirus is present in a nonreplicating form in myelomonocytic progenitor cells6,11,12,16,33,35 and stromal fibroblasts,36 integration of FeLV provirus in latent infection may alter function of these cell and contribute to the pathogenesis of myelosuppressive disorders. Through integration of proviral DNA into the genome of the host cell, regulatory mechanisms may be affected, and myelogenesis may be disturbed. Alternatively, FeLV provirus could cause bone marrow disorders by inducing the expression of unknown antigens on the cell surface that result in immune-mediated destruction of the cell. Nonregenerative cytopenia and latent FeLV infection also could be unrelated and merely appear in the same cat by chance.

One limitation of the present study is that certain hematologic abnormalities, including anemia of chronic disease or immune-mediated nonregenerative anemia, could not be excluded in the patient group. Furthermore, not every diagnostic test was performed in all of the cats. In addition, the inclusion criteria for nonregenerative neutropenia were very strict (segmented neutrophil counts < 2,500/μL and bands <400/μL).

The results of this study suggest that FeLV latency does not play an important role in cats with nonregenerative cytopenias. Although atypical infections have been described in which FeLV is not detectable in bone marrow but in other tissues including, spleen, lymph node, and small intestines,41 it is unlikely that this was the case in our study because FeLV-related nonregenerative cytopenia can only be explained by the presence of at least proviral DNA if not the entire replication-competent virus.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

aFeline Leukemia Virus Antigen/Feline Immunodeficiency Virus Antibody Test Kit; IDEXX, Westbrook, ME

bAllegiance, Deerfield, IL

cBecton Dickinson Labware, Becton Dickinson and Company, Franklin Lakes, NJ

dQIAGEN Inc, Valencia, CA

eSynbiotics, San Diego, CA

fMagNA Pure; F. Hoffmann-La Roche AG, Basel, Switzerland

gCAS 1200; Corbett Life Science, Mortlake, NSW, Australia

hDUNN Labortechnik, Ansbach, Germany

iDAKO, Hamburg, Germany

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

We thank Dr Carola Sauter-Louis (Clinic for Ruminants, LMU University of Munich, Germany) for her support in the statistical evaluation.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
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