T-lymphocyte profiles in FIV-infected wild lions and pumas reveal CD4 depletion.
ABSTRACT Feline immunodeficiency virus (FIV) is a lentivirus related to human immunodeficiency virus (HIV) that causes feline AIDS in the domestic cat (Felis catus). Serological surveys indicate that at least 25 other species of cat possess antibodies that cross-react with domestic cat FIV. Most infected nondomestic cat species are without major symptoms of disease. Long-term studies of FIV genome variation and pathogenesis reveal patterns consistent with coadaptation of virus and host in free-ranging FIV-Ple-infected African lions (Panthera leo) and FIV-Pco-infected pumas (Puma concolor) populations. This report examined correlates of immunodeficiency in wild and captive lions and pumas by quantifying CD5(+), CD4(+), and CD8(+) T-cell subsets. Free-ranging FIV-Ple-infected lions had immunofluorescence flow cytometry (IFC) profiles marked by a dramatic decline in CD4(+) subsets, a reduction of the CD4(+)/CD8(+) ratio, reduction of CD8(+)beta(high) cells, and expansion of the CD8(+)beta(low) subset relative to uninfected lions. An overall significant depletion in CD5(+) T-cells in seropositive lions was linked with a compensatory increase in total CD5(-) lymphocytes. The IFC profiles were altered significantly in 50% of the seropositive individuals examined. The FIV-Pco-infected pumas had a more generalized response of lymphopenia expressed as a significant decline in total lymphocytes, CD5(+) T-cells, and CD5(-) lymphocytes as well as a significant reduction in CD4(+) T-cells. Like lions, seropositive pumas had a significant decline in CD8(+)beta(high) cells but differed by not having compensatory expansion of CD8(+)beta(low) cells relative to controls. Results from FIV-infected lions and pumas parallel human and Asian monkey CD4(+) diminution in HIV and SIV infection, respectively, and suggest there may be unrecognized immunological consequences of FIV infection in these two species of large cats.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: A high percentage of free-ranging pumas (Felis concolor) are infected with feline lentiviruses (puma lentivirus, feline immunodeficiency virus Pco [FIV-Pco], referred to here as PLV) without evidence of disease. PLV establishes productive infection in domestic cats following parenteral exposure but, in contrast to domestic cat FIV, it does not cause T-cell dysregulation. Here we report that cats exposed to PLV oro-nasally became infected yet rapidly cleared peripheral blood mononuclear cell (PBMC) proviral load in the absence of a correlative specific immune response. Two groups of four specific-pathogen-free cats were exposed to PLV via the mucosal (oro-nasal) or parenteral (i.v.) route. All animals were PBMC culture positive and PCR positive within 3 weeks postinfection and seroconverted without exhibiting clinical disease; however, three or four oro-nasally infected animals cleared circulating proviral DNA within 3 months. Antibody titers reached higher levels in animals that remained persistently infected. PLV antigen-induced proliferation was slightly greater in mucosally inoculated animals, but no differences were noted in cytotoxic T-lymphocyte responses or cytokine profiles between groups. The distribution of virus was predominantly gastrointestinal as opposed to lymphoid in all animals in which virus was detected at necropsy. Possible mechanisms for viral clearance include differences in viral fitness required for crossing mucosal surfaces, a threshold dose requirement for persistence, or an undetected sterilizing host immune response. This is the first report of control of a productive feline or primate lentivirus infection in postnatally exposed, seropositive animals. Mechanisms underlying this observation will provide clues to containment of immunodeficiency disease and could prompt reexamination of vaccine-induced immunity against human immunodeficiency virus and other lentiviruses.Journal of Virology 04/2005; 79(5):2797-806. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: While viral infections and their impact are well studied in domestic cats, only limited information is available on their occurrence in free-ranging lions. The goals of the present study were (i) to investigate the prevalence of antibodies to feline calicivirus (FCV), herpesvirus (FHV), coronavirus (FCoV), parvovirus (FPV), and immunodeficiency virus (FIV) and of feline leukemia virus (FeLV) antigen in 311 serum samples collected between 1984 and 1991 from lions inhabiting Tanzania's national parks and (ii) to evaluate the possible biological importance and the interrelationship of these viral infections. Antibodies to FCV, never reported previously in free-ranging lions, were detected in 70% of the sera. In addition, a much higher prevalence of antibodies to FCoV (57%) was found than was previously reported in Etosha National Park and Kruger National Park. Titers ranged from 25 to 400. FeLV antigen was not detectable in any of the serum samples. FCoV, FCV, FHV, and FIV were endemic in the Serengeti, while a transient elevation of FPV titers pointed to an outbreak of FPV infection between 1985 and 1987. Antibody titers to FPV and FCV were highly prevalent in the Serengeti (FPV, 75%; FCV, 67%) but not in Ngorongoro Crater (FPV, 27%; FCV, 2%). These differences could be explained by the different habitats and biological histories of the two populations and by the well-documented absence of immigration of lions from the Serengeti plains into Ngorongoro Crater after 1965. These observations indicate that, although the pathological potential of these viral infections seemed not to be very high in free-ranging lions, relocation of seropositive animals by humans to seronegative lion populations must be considered very carefully.Clinical and Diagnostic Laboratory Immunology 10/1996; 3(5):554-62. · 2.51 Impact Factor
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ABSTRACT: An African lioness from the Zoo of Zurich had to be euthanized because of an inoperable tumor. The serum tested negative for feline leukemia virus (FeLV) p27 antigen by enzyme-linked immunosorbent assay (ELISA) but was strongly positive for feline immunodeficiency virus (FIV) antibodies by ELISA and Western blot. When her only offspring and mate were tested for FIV, high antibody titers to FIV were also found in their serum. Lymphocytes were prepared from these two lions on different occasions and co-cultivated with specific pathogen free (SPF) cat lymphocytes in the presence of concanavalin A and recombinant human interleukin-2 (IL-2) for 6 weeks. The cell culture supernatants tested negative for Mg(2+)-dependent reverse transcriptase and FIV p24 by a double antibody sandwich ELISA throughout the culture period. Whole blood and buffy coat cells collected from these two lions were transmitted by intraperitoneal injection into two SPF cats. The two cats did not seroconvert for a period of 11 months nor could reverse transcriptase activity and FIV p24 antigen be demonstrated in the supernatant of several lymphocyte cultures. To determine the importance of lentivirus infections in zoo-kept wild felids, 124 serum samples were obtained from African lions, Indian and Siberian tigers, snow leopards, panthers, cheetahs and other wild cats from nine European zoos. In addition, serum samples collected from 12 Asiatic lions originating from Gir forest in the Indian State of Gujarat were included in this study. The sera were tested for antibodies to FIV, FeLV and feline syncytium-forming virus (FeSFV) by ELISA and Western blot using the respective viruses after gradient purification. In addition, some of the sera were also tested for antibodies to equine infectious anemia virus (EIAV) and Visna-Maedi virus (VMV). Antibodies to FIV were found in 30/53 (57%) of African lions, one of 18 tigers and one of four panthers. All other sera including those collected from the 12 Asiatic lions were negative for FIV antibodies. Some of the FIV positive lion sera had high antibody titers producing strong bands on Western blot strips even in dilutions of > 1:1000. The Western blot pattern of the lion sera differed from that of domestic cats in that primarily p24 and to a lesser degree p17 was recognized. Antibodies to FeSFV were found in 14 animals (seven with strong, seven with intermediate, reaction). No correlation was found between FIV and FeSFV infection. Antibodies to FeLV were found in two cheetahs which later turned out to have been vaccinated with Leukocell, a FeLV vaccine.(ABSTRACT TRUNCATED AT 400 WORDS)Veterinary Immunology and Immunopathology 01/1993; 35(1-2):215-24. · 1.88 Impact Factor
T-LYMPHOCYTE PROFILES IN FIV-INFECTED WILD LIONS AND
PUMAS REVEAL CD4 DEPLETION
M. E. Roelke,1J. Pecon-Slattery,2,8S. Taylor,3S. Citino,4E. Brown,2,7C. Packer,5
S. VandeWoude,6and S. J. O’Brien2
1Laboratory of Genomic Diversity, Basic Research Program, SAIC Frederick, National Cancer Institute, Frederick,
Maryland 21702, USA
2National Cancer Institute–Frederick, Frederick, Maryland 21702, USA
3Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA
4White Oak Conservation Center, Yulee, Florida 32097, USA
5Department of Ecology, Evolution and Behavior, University of Minnesota, St. Paul, Minnesota 55108, USA
6Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523, USA
7Present address: U.S. Food and Drug Administration, College Park, Maryland 20740, USA
8Corresponding author (email: email@example.com)
ciency virus (HIV) that causes feline AIDS in the domestic cat (Felis catus). Serological surveys
indicate that at least 25 other species of cat possess antibodies that cross-react with domestic cat
FIV. Most infected nondomestic cat species are without major symptoms of disease. Long-term
studies of FIV genome variation and pathogenesis reveal patterns consistent with coadaptation of
virus and host in free-ranging FIV-Ple–infected African lions (Panthera leo) and FIV-Pco–infected
pumas (Puma concolor) populations. This report examined correlates of immunodeficiency in wild
and captive lions and pumas by quantifying CD5+, CD4+, and CD8+ T-cell subsets. Free-ranging
FIV-Ple–infected lions had immunofluorescence flow cytometry (IFC) profiles marked by
a dramatic decline in CD4+ subsets, a reduction of the CD4+/CD8+ ratio, reduction of CD8+bhigh
cells, and expansion of the CD8+blowsubset relative to uninfected lions. An overall significant
depletion in CD5+ T-cells in seropositive lions was linked with a compensatory increase in total
CD52 lymphocytes. The IFC profiles were altered significantly in 50% of the seropositive
individuals examined. The FIV-Pco–infected pumas had a more generalized response of
lymphopenia expressed as a significant decline in total lymphocytes, CD5+ T-cells, and CD52
lymphocytes as well as a significant reduction in CD4+ T-cells. Like lions, seropositive pumas
had a significant decline in CD8+bhighcells but differed by not having compensatory expansion
of CD8+blowcells relative to controls. Results from FIV-infected lions and pumas parallel human
and Asian monkey CD4+ diminution in HIV and SIV infection, respectively, and suggest there
may be unrecognized immunological consequences of FIV infection in these two species of large
CD4 T-cells, Felidae, FIV, flow cytometry, immune depletion, lion, lympho-
Feline immunodeficiency virus (FIV) is a lentivirus related to human immunodefi-
Feline immunodeficiency virus (FIV) is
a pathogenic lentivirus related to human
immunodeficiency virus (HIV) and simian
immunodeficiency virus (SIV). Initially
isolated in 1986, FIV was subsequently
identified as the etiologic agent of ac-
quired immune deficiency syndrome or
feline AIDS in the domestic cat (Felis
catus) (Pedersen et al., 1987; Brunner and
Pedersen, 1989; Pedersen et al., 1989;
Gardner, 1991). Feline immunodeficiency
virus–infected domestic cats exhibit dis-
ease symptoms, immune suppression, and
mortality markedly similar to HIV in-
fection in humans (Yamamoto et al.,
1988; Pedersen et al., 1989; Yamamoto et
al., 1989; Willett et al., 1993; English et al.,
1994; Bendinelli et al., 1995; Liang et al.,
2000). Feline immunodeficiency virus
strains are species-specific as indicated
by comparative genomic analyses of
strains sequenced from domestic cats
(FIV-Fca), Pallas cats (Otocolobus manul;
FIV-Oma), cheetahs (Acinonyx jubatus;
FIV-Aju), leopards (Panthera pardus;
FIV-Ppa), pumas (Puma concolor; FIV-
Pco), and African lions (Panthera leo; FIV-
Ple) (Brown et al., 1994; Carpenter et al.,
1996, 1998; Troyer et al., 2005). Additional
serological surveys indicate at least 25
Journal of Wildlife Diseases, 42(2), 2006, pp. 234–248
#Wildlife Disease Association 2006
other species of cats possess antibodies
that cross-react with FIV (Olmsted et al.,
1992; Brown et al., 1994; Carpenter and
O’Brien, 1995; Carpenter et al., 1996,
1998; Troyer et al., 2004, 2005). The
observed worldwide prevalence of FIV in
multiple cat species is made more in-
triguing by the apparent lack of discern-
able disease in nondomestic cat species
(Lutz et al., 1992; Hofmann-Lehmann et
al., 1996; Packer et al., 1999), although an
FIV-positive captive lion with end-stage
disease reminiscent of domestic cats with
feline AIDS has been reported (Poli et al.,
1995; Bull et al., 2003).
Phylogenetic studies of the gag and pol
genes from FIV in natural populations of
pumas in North and South America and
African lions in eastern and southern
Africa and env regions from FIV-Fca
sampled from domestic cats worldwide
depict unique patterns of virus-host co-
evolution. In pumas, FIV-Pco forms dis-
tinct divergent evolutionary lineages that
are distributed throughout this species
range (Carpenter et al., 1996; Biek et al.,
2003). In lions, FIV-Ple is endemic to
populations within east and south Africa
and forms at least three different evolu-
tionary clades with high levels of genetic
differences (Brown et al., 1994; Troyer et
al., 2004, 2005). Genetic analyses of FIV-
Ple within a large out-bred population of
lions in the Serengeti region of Tanzania
revealed .90% prevalence with 43% of
the individuals multiply infected with at
least two subtypes (Brown et al., 1994;
Troyer et al., 2004). In domestic cats, FIV-
Fca subtype classification is made using
the more variable env region rather than
the pol or gag analyzed in FIV from other
species. Even using the env region, FIV-
Fca has lower levels of intersubtype
divergence than FIV-Pco and FIV-Ple
(Carpenter et al., 1998). Further, three
of the five recognized subtypes or clades
of FIV-Fca are composed of closely re-
lated strains from cats dwelling on differ-
ent continents (Carpenter et al., 1998).
Thus, the low genetic diversity and de-
monstrable pathogenicity of FIV in do-
mestic cats suggest that this species
acquired FIV relatively recently, whereas
FIV-Pco and FIV-Ple descend from an
earlier species experience with endemic
virus and became attenuated as a natural
outcome of a longer period of virus-host
coevolution (Carpenter and O’Brien,
1995; Carpenter et al., 1996, 1998).
Under this scenario, FIV-Fca, FIV-Ple,
and FIV-Pco would be expected to elicit
different clinicopathologic abnormalities
in their respective host species. However,
evaluation of such parameters using a sys-
tematic approach in lions and pumas has
been impeded by logistical difficulties and
infrequent opportunities associated with
sample collection in the wild, adequate
preservation of samples in field conditions,
and lack of reagents or reagent validation
to measure blood cell responses for exotic
felids accurately. Consequently, only one
previously published study has reported
lymphocyte subset alterations in five
captive FIV-Ple seropositive lions (Bull
et al., 2003) Additionally, the high ser-
oprevalence rate of some free-ranging
populations (up to 100%) (Brown et al.,
1994; Carpenter and O’Brien, 1995; Biek
et al., 2003; Troyer et al., 2004) may affect
statistical analyses of seropositive versus
Analysis of domestic cats infected with
FIV-Fca, on the other hand, has been
more intensively studied. Domestic cats
infected with FIV-Fca experience pro-
found changes in T-cell subsets concur-
rent with clinical immune deficiency. Like
HIV in humans, the continued deteriora-
tion of the host immune system in FIV-
positive domestic cats is correlated with
the reduction in circulating CD4+ lym-
phocytes (Ackley et al., 1990; Novotney et
al., 1990). In the initial acute phase, the
CD4+/CD8+ ratio is lowered as a conse-
quence by both the reduction of CD4+ T-
cells and the marked expansion of CD8+
T-cells (Willett et al., 1993). However,
subtype and/or strain-specific effects on
the host circulating lymphocyte kinetics,
ROELKE ET AL.—T-LYMPHOCYTE PROFILES IN LIONS AND PUMAS235
which parallels the degree of immunosup-
pression or virulence, are observed in
domestic cats infected with a less virulent
strain of FIV characterized by low viral
load (Hosie et al., 2002). Experimental
infection of domestic cats with FIV-Pco or
FIV-Ple results in apathogenic but pro-
ductive infection (VandeWoude et al.,
1997a, b, 2003; Terwee et al., 2005),
which strengthens the hypothesis that
nondomestic cat FIVs are host-adapted
or perhaps less virulent lentiviruses.
We examined changes in T-cell lym-
phocytes in response to FIV infection in
a cohort of both captive and free-ranging
populations of lions and pumas and found
significant changes in circulating lympho-
cyte T-cell subsets associated with FIV
infection. These findings may have signif-
icant implications for management of FIV-
positive endangered feline species and for
the health of individual animals.
Study animals: pumas and lions
The FIV-positive and -negative individ-
uals were sampled from pumas in North
America and African lions from the
Serengeti National Park in Tanzania and
Kruger National Park in South Africa
(Table 1). All pumas and free-ranging
African lions were captured, anesthetized,
examined, and bled as part of ongoing
field studies (Roelke et al., 1993; Roelke-
Parker et al., 1996). The puma sample
consisted of 10 uninfected individuals (1–
9 yr-old) and six FIV-Pco–infected ani-
mals (5–12 yr-old); five were captive, and
11 were free-ranging, all originating from
the same population in southern Florida,
USA. Twelve FIV-Ple–infected lions (2–
12 yr-old) and five zoo-bred captive naive
lions of unknown ages with no record of
ancestral geographic origin were sampled.
All seropositive lions were free-ranging in
either Kruger National Park, South Africa,
or Serengeti National Park, Tanzania.
Captive lions were bled during routine
yearly physical examinations.
Sample collection and processing
Peripheral whole blood collected from
both captive and free-ranging animals was
processed within 48 hr. Whole blood
collected with EDTA was used for an
absolute white blood cell count (WBC),
determined by the UnopipetteH counting
system and hemocytometer, and an abso-
lute lymphocyte count, determined by
staining blood films with a modified
Wright-Giemsa stain. To process samples
for lymphocyte analyses, peripheral blood
mononuclear cells (PBMCs) were sepa-
rated from whole anticoagulated blood
collected by using Histopaque-1077 (Sig-
ma, St. Louis, Missouri, USA) and viably
frozen. Five milliliters of whole blood was
overlaid on 5 ml histopaque and spun at
400 3 G for 30 min. The PBMC layer was
removed, washed in phosphate buffered
saline (PBS), and spun at 250 3 G for
10 min at least twice. Cell pellets were
gently suspended in 90% fetal calf serum
with 10% dimethyl sulfoxide (DMSO) and
viably frozen, in aliquots of 107cells/ml/
cryotube, at a rate of 1 C/min and stored
in liquid nitrogen.
Serum samples were simultaneously
collected and screened for antibodies to
both FIV-Pco and FIV-Ple antigens.
Western blot analysis for FIV-reacting
antibodies was performed as previously
described (Diehl et al., 1995; Vande-
Woude et al., 1997a, b; Troyer et al.,
2005). Tissue culture supernatant contain-
ing either virus strain grown on 3201
T-cells was concentrated by ultracentrifu-
gation. Protein content was determined,
and viral antigen was subjected to poly-
acrylamide electrophoresis and trans-
ferred to nylon membranes. Diluted
serum was reacted with membrane strips,
and bound antibody detected by horse
radish peroxidase-labeled secondary anti-
bodies. To ensure FIV antibody detection,
FIV-Fca antigen was used in separate tests
in addition to FIV-Pco for pumas and
FIV-Ple for lions. Positive and negative
sera served as controls for comparison to
236JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006
TABLE 1. Species, identity, health status, origin and contact for samples used in this study.
Puma (Puma concolor)
PCO-1668 Aug 1991
M5 Captive HealthyEverglades Holiday
Big Cypress Swamp,
Big Cypress Swamp,
Big Cypress Swamp,
Big Cypress Swamp,
Big Cypress Swamp,
Big Cypress Swamp,
White Oak Planta-
PCO-387 6 Aug 1991
M9Captive HealthyM. Roelke
PCO-390 6 Aug 1991
F8 CaptiveHealthyM. Roelke
PCO-3927 Aug 1991
M7Captive Healthy M. Roelke
PCO-533 16 Dec 1996 2
M5 Wild Healthy S. Taylor/D.
PCO-539 15 Jan 1997
PCO-717 8 Mar 1997
PCO-718 18 Mar 1997 2
PCO-719 15 Apr 1997 2
PCO-898 5 Mar 1997
PCO-075 14 Jan 1997
F 12Captive Anemia,
Healthy PCO-154 25 Jan 1992
M5WildBig Cypress Swamp,
Big Cypress Swamp,
Big Cypress Swamp,
Big Cypress Swamp,
Big Cypress Swamp,
PCO-160 21 Feb 1997
F 10Wild Mange
PCO-730 18 Dec 1996
PCO-733 19 Dec 1996
PCO-736 10 Jan 1997
African lion (Panthera leo)
PLE-030 5 Feb 1997
M3Captive HealthyWildlife Waystation,
Druid Park Zoo, Bal-
Kruger National Park,
Kruger National Park,
Kruger National Park,
Kruger National Park,
PLE-031 28 Feb 1997 2
M n/aCaptiveHealthyR. Yates
PLE-032 28 Feb 1997 2
PLE-0337 Mar 1997
F9 CaptiveAsthmaM. Cranfield
PLE-0341 Apr 1997
PLE-133 26 July 1992
PLE-134 27 July 1992
PLE-135 26 July 1992
PLE-138 27 July 1992
ROELKE ET AL.—T-LYMPHOCYTE PROFILES IN LIONS AND PUMAS237
Immunofluorescence flow cytometry
Viably frozen PBMCs were quickly
thawed at 37 C, washed in PBS, counted,
and resuspended in a buffer solution of
PBS. The PBMCs (13106) were com-
bined in separate tubes with monoclonal
antibodies that recognize the following:
feline CD4+ (Fel7 clone CD4; Klotz and
Cooper, 1986; Ackley et al., 1990); feline
CD8+ (FT2 clone fCD8-Beta; Klotz and
Cooper, 1986); and feline Pan-T+ cell
CD5 equivalent (clone f43; Ackley and
Cooper, 1992). The T-cell profiles were
generated by double labeling with CD5+/
CD4+, CD5+/CD8+, or CD4+/CD8+ and
quantified by two-color immunofluores-
cence flow cytometry (IFC) using Becten-
Dickenson FACscan and Cell Quest
software. A dot-plot of side and forward
scatter was used to construct a live lym-
phocyte gate of 5,000–10,000 cells per
assay. The absolute number of T-cells
was determined by multiplying the frac-
tion of respective CD4+, CD8+, and
CD5+ subpopulations times the number
of lymphocytes/ml within the original
sample. The CD52 cells, within the gated
lymphocyte fraction, were considered
primarily B cells, though it is possible that
this fraction could also contain small
proportions of monocytic cells. No con-
version values were available for lions
sampled from Kruger Park (Ple 133, 134,
135, 138). The potential bias introduced
by using viably frozen PBMC versus those
from fresh blood for IFC was addressed
by setting the gates to exclude nonspecific
binding and thus compensate for back-
ground staining due to cell death. More-
over, IFC analysis provided comparable
results whether performed on frozen or
fresh PBMC in our experience (Vande-
Woude, unpublished data) as well as that
of others (Bull et al., 2003).
PLE-569 28 June 1994
F3Wild Chronic CDV
Acute CDV infec-
PLE-5861 Apr 1994
PLE-599 20 July 1994
F13 WildSerengeti National
PLE-605 19 July 1995
PLE-6187 July 1994
F4Wild Prior CDV infec-
PLE-645 18 Mar 1995
F 8 to 10Wild Serengeti National
PLE-647 10 July 1995
M 6 to 8 WildSerengeti National
PLE-64917 Jan 1996
M 6 to 8 Wild Serengeti National
aFIV status determined by Western blot (see Methods).
bRemoved from the wild in 1987.
238 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006
The total T-cell (CD5+) profile gener-
ated by IFC displayed the relative propor-
tions of CD4+, CD8+, and CD42CD82
subpopulations within the PBMC sample.
These ratios were not independent be-
cause they were derived from the total
CD5+ T-cells; therefore, we performed
heterogeneity G-tests (Sokol and Rohlf,
1991). Each profile was tested against the
expected profile from control naive ani-
mals to determine if the individual T-cell
subset profile changed with FIV infection.
Significance was determined by computa-
tion of a G-statistic that approximates a chi
For each IFC profile, the relative
proportions of CD4+, CD8+, and CD42
CD82CD5+ cells were converted into
absolute numbers of cells based on the
absolute cell count of lymphocytes. We
evaluated the following absolute cell
counts for each animal: WBC/ml, total
lymphocytes/ml, total CD52 cells/ml,
CD5+ T-cells/ml, CD4+/ml, CD8+/ml,
and CD42CD82CD5+/ml. In addition,
we examined two subsets, CD8+blowand
CD8+bhigh, identified by the antibody FT2
as it binds specifically to the beta chain of
the CD8+ heterodimer molecule. Esti-
mates of cell counts for each were com-
puted by multiplying the relative pro-
portion times the estimated CD8+ cells/
ml count for each individual.
The distribution of each cell count
variable was tested for normality using
the Shapiro-Wilks’ W suitable for small
sample sizes. The relative impact of FIV
status on the cell count variables for each
species was tested using analysis of
variance (ANOVA) and paired t-tests of
means (SAS, 2001).
The majority of lymphocytes of 17 lion
and 16 puma blood samples were CD5+
T-cells by IFC. The relative proportion of
CD4+, CD8+, and CD42CD82 subsets
among CD5+ T-lymphocytes were de-
termined and presented as a CD5+ T-cell
profile per animal (Table 2). Further, cell
count data were used to test for changes in
WBC/ml, total lymphocytes/ml, total
CD52 cells/ml, CD5+ T-cells/ml, CD4+
cells/ml, CD8+ T-cells/ml, CD8bhigh/ml,
cells/ml. Shapiro-Wilks’ W test indicated
normal distributions for each of these cell
count variables. Two of these variables,
WBC/ml and CD8+ T-cells/ml, did not
change with FIV status in either lions or
pumas (Table 3; Figs. 1a, b).
T-cell alterations with FIV infection in pumas
The IFC profiles of the relative propor-
tions of CD4+, CD8+, and CD42CD82
CD5+ cells within FIV-negative pumas
were uniform among individuals (G-test,
NS). The mean T-cell profile for pumas
infected with FIV-Pco was 42:27:29 (per-
centage of CD4+, CD8+, and CD42
CD82CD5+, respectively). Three of the
six FIV-Pco–infected pumas (Pco-075,
Pco-733, and Pco-736) had significant
differences in IFC profiles (Table 2), but
the overall mean profile for positive
animals was not significantly altered
relative to negative controls (G-test,
Infection with FIV-Pco was correlated
with an overall reduction in cell counts
of total lymphocytes (t-test, P50.03) that
involved both CD5+ T-cells and CD52
lymphocyte subsets and was accompa-
nied by a 50% reduction in CD4+ cells/
ml relative to uninfected pumas (t-test,
P50.008) (Table 3 and Fig. 1a). No
significant changes were observed in
the CD8+ and CD42CD82CD5+ T-
cell subsets relative to the negative
controls (Tables 2 and 3). Further exam-
ination of the CD8+ T-cell subset
revealed no change in CD8+blowcells,
CD8+bhighcells was detected with FIV-
Pco infection (t-test, P5 0.008) (Table 3
and Fig. 2a, b).
ROELKE ET AL.—T-LYMPHOCYTE PROFILES IN LIONS AND PUMAS239
T-cell alterations with FIV infection in lions
Homogeneous IFC profiles of uninfect-
ed lion controls had an average ratio of
60 : 22 : 18 for relative CD4+, CD8+, and
CD42CD82CD5+ percentages. In con-
trast, IFC profiles for infected lions were
P59.031029), and each had significant
deviation from the mean profile (G-test,
P52.531025) (Table 2).
Measured cell counts indicated total
lymphocytes did not vary with FIV-Ple
infection as observed with FIV-Pco in
pumas (Table 3 and Fig. 1b). Instead,
infected lions had both a significant
Immunofluorescent profiles of lymphocytes in FIV infected pumas and lions and uninfected
Species ID FIV
Proportion of CD5+
Proportion of CD8b+ (%)Ratio
Puma (Puma concolor)
African lion (Panthera leo)
aDenotes individuals with significantly different IFC profile from mean profile of control animals.
240JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006
Absolute cell counts standardized by total T-cell counts (see Methods, Table 2).
Absolute cell counts (103/ml)
Estimated Cell Counts (103/ml)
Puma (Puma concolor)
LION (Panthera leo)
ROELKE ET AL.—T-LYMPHOCYTE PROFILES IN LIONS AND PUMAS 241
P50.0025) and a dramatic 80% decline in
CD4+ cells (t-test, P50.0001) relative to
controls (Fig. 1b). This CD4+ cell decline
was observed without a significant in-
crease in overall CD8+ levels. However,
within the CD8+ subset, bhighcells de-
clined precipitously with FIV-Ple infection
to one-third the levels observed in controls
(t-test, P50.006) and was accompanied by
an expansion of blowcells (t-test, P50.03)
(Fig. 2a, b). Further unique changes were
observed including an increase in the cell
counts of the CD42CD82CD5+ subset
(t-test, P50.05) and an increase in the
CD52 lymphocytes (t-test, P50.001),
presumed to be mostly B cells, in infected
lions relative to negative controls (Table 3
and Fig. 1b).
Alteration of the CD4+/CD8+ ratio with FIV infection
Both lions and pumas were examined
for reduction of the CD4+/CD8+ ratio
commonly observed in FIV-Fca infection
of domestic cats. In lions, the CD4+/
mean52.95 (range of 2.23–5.04) in con-
trols to mean51.17 (range of 0.21–2.57) in
FIV-Ple–infected individuals (Table 2). In
contrast, the puma CD4+/CD8+ T-cell
ratio was not significantly altered (Ta-
ble 2) with values slightly less in pumas
with FIV-Pco mean51.73 (range of 0.91–
2.57) compared with mean51.93 (range of
1.37–2.92) in negative animals. The drop
in CD4+ cells in positive pumas was
accompanied by a proportional, albeit
not significant, reduction in CD8+ cells
(Fig. 1b) sufficient to maintain the CD4+/
CD8+ ratio as unchanging with FIV-Pco
To understand the interaction between
FIV and target cells in the host immune
system, we examined T-lymphocyte pro-
files in free-ranging and captive lions and
pumas naturally infected with species-
specific strains FIV-Ple and FIV-Pco,
respectively. The uninfected controls of
the two species had only minor differences
in the relative mean proportions of
subsets (51:27:22 in pumas; 60:22:18 in
lions). However, FIV infection appeared
to affect lions and pumas differently,
causing marked changes in lymphocytes
unique to each species. The T-cell subset
profile perturbations were more pro-
nounced in lions than pumas with FIV
infection. Specifically, all seropositive
lions, but only 50% of FIV-infected
pumas, had significantly altered IFC
profiles relative to negative controls.
The major alteration in the relative
proportion and absolute cell count of
CD4+, CD8+, and CD42CD82 subsets
in both seropositive lions and pumas was
a reduction in CD4+ T-cells. In domestic
cats infected with FIV-Fca, the CD4+/
CD8+ ratio declines during the asymp-
tomatic phase from two or more to less
than one (English et al., 1994; Bendinelli
et al., 1995;) because of CD4+ depletion
concurrent with relative or absolute CD8+
increase. The magnitude of CD4+ de-
pletion was profound in FIV-Ple lions and
resulted in a sharp decline in the CD4+/
CD8+ ratio. By contrast, the CD4+/CD8+
ratio was not significantly altered in pumas
even though FIV-Pco infection was corre-
lated with a 50% decline in CD4+ T-cells.
Additional differences between lions
and pumas included alterations of total
lymphocytes and CD5+ T-cells; neither of
these changes paralleled the typical find-
ings in domestic cats. For example, pumas
infected with FIV-Pco had relative lym-
phopenia, indicated by the 41% lower
total lymphocyte count, which was distrib-
uted equally between CD5+ and CD52
cell subsets. Although lymphopenia was
not noted in seropositive lions (Fig. 1b),
a significant decrease (48%) in CD5+ T-
cells was accompanied by a compensatory
increase in CD52 lymphocytes that
masked this loss. Most studies have not
recorded lymphopenia or pan-T-cell loss
as a striking finding in domestic cat FIV
infection. Therefore, assuming CD52
242JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006
cells are predominantly B-cells, this in-
crease would be a unique feature to FIV-
Ple infection as B-cell lymphocyte kinetics
in FIV-Fca infected cats have not been
reported to be altered relative to naive
controls (Ackley et al., 1990; Novotney et
Reduction in CD4+ cells in lions and
pumas was not accompanied by an abso-
lute increase of CD8+ cells as observed in
domestic cats. During the acute and
asymptomatic phases of FIV infection,
CD8+ T-cells are both cytotoxic and
virus-suppressive in domestic cats (Prince
et al., 1991; English et al., 1994). In
particular, the heterodimer CD8+ mole-
cule exhibits changes in the composite
a and b chains correlated with time course
of viral infection, antiviral activity, and
pathogenesis of disease (Shimojima et al.,
1998). The CD8+ subset changes in lions
and pumas in this study were based on the
FT-2 antibody that binds exclusively to the
b chain of the heterodimer. Previous
studies of the CD8+ b chain in the
asymptomatic phase of FIV infection in
CD8+bhighcells and the appearance and
expansion of CD8+blowT cells, which
secrete a soluble factor inhibitory for in
vitro FIV infection (Bucci et al., 1998a, b;
Shimojima et al., 1998; Gebhard et al.,
1999). This simultaneous expansion of
CD8+blowT-cell subsets offsets a decrease
in total CD5+ T-cells with FIV-Fca in-
fection (Willett et al., 1993). In this study,
lions and pumas resembled domestic cats
by having a significant depletion of
cells and a reduction of
CD5+ cells, but only lions had a concom-
itant expansion of CD8+blowcells. A
similar result in a study of five seropositive
captive lions with an expanded CD8+blow
subset relative to naive animals (Bull et al.,
2003) suggests this is a signature of FIV-
Ple infection. Results from FIV-Pco–
infected pumas demonstrated a decline
in lymphocytes in general, and CD4+ cells
in particular, which was not offset by
expansion of any subset presented here.
Rather, a substantial number of CD8+blow
cells are present in pumas irrespective of
FIV status. Perhaps these cells are part of
an immune response to another unspeci-
fied pathogen, or this may reflect the
outcome of coevolution of virus and host
that has resulted in a ‘‘standing army’’
within the species as a whole.
The changes in CD4+ subpopulations in
lions and to a lesser extent in pumas in
response to FIV infection provide strong
support for T-cell dyscrasia as observed
with domestic cats. Yet other lymphocyte
changes specific to lions and pumas
suggest that additional immune responses
occur in both species, and perhaps clues
reside within an uncharacterized T-cell
population present within the CD42
CD82 subset. Although pumas showed
no change in CD42CD82 cell counts
with FIV-Pco infection, FIV positive lions
had a significant increase relative to
controls. Thus, the more muted immune
response of pumas consisting of general
lymphopenia and reduction of CD4+ cells
not accompanied by a change in the
causes less T-cell perturbation in pumas
than FIV-Ple in lions. The FIV-Ple elicits
a dramatic decline in CD4+ cells, yet lions
may be in the process of coadapting to the
virus, as suggested by the unique combi-
nation of increased CD52 cells, expansion
of the CD8+blowsubset, and increased
numbers of CD42CD82 cells.
Alternatively, extreme changes in CD4+
levels in lions also may be influenced by
other determinants such as length of FIV
infection or other microbial infections, as
well as relative binding affinities of mono-
clonal antibodies originally developed for
domestic cat T-cell antigens. The highly
heterogeneous IFC profiles observed
among individual wild seropositive lions
relative to the more uniform profiles of
control lions in zoos might reflect stress
differences between captive and free-
ranging populations. The fact that FIV
status increases with age, and is seen
with high seroprevalence in many free-
ROELKE ET AL.—T-LYMPHOCYTE PROFILES IN LIONS AND PUMAS243
244JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006
ranging populations of lions (Brown et al.,
1994; Troyer et al., 2004), also makes it
difficult to determine how age and envi-
ronmental conditions may have contribut-
edtoour findings. Wewereunabletoassess
the effect of age in this study because of
insufficient replicates per age class and
unknown ages for some of both positive and
animals. Boxed regions reflect 25th–75th percentile range of individual values. Bars represent 10th–90th
percentile range. Means are plotted as white line within boxes. P-values are t-test of mean between FIV
positive (puma n56; lion n 5 12) and negative (puma n510; lion n55) animals within each species:
(a) CD8+bhighin pumas and lion, (b) CD8+blowin lions and pumas.
Comparison of CD8+ subsets bhighand blowestimated cell counts in FIV-positive and -negative
cells, total CD5+ T-cells, and T-cell subpopulations from whole blood between FIV-positive and FIV-negative
individuals. (a) Puma positive n56; puma negative n510. (b) Lion positive n512; lion negative n55.
Comparison of mean cell counts (3103/ml) of absolute lymphocytes and estimated total CD52
ROELKE ET AL.—T-LYMPHOCYTE PROFILES IN LIONS AND PUMAS 245
negative lions, leading to a prohibitively
small sample for statistical analysis.
Overall, this immunological survey of
naturally FIV-infected animals may serve
as an indictor of the susceptibility of both
captive and wild populations to emerging
disease. Outbreaks of opportunistic infec-
tions, each with varying degrees of path-
ogenicity, have been documented in non-
domestic species of cat. In lions, a canine
distemper virus (CDV) outbreak caused
significant mortality in free-ranging FIV-
infected African lions of the Serengeti
ecosystem in 1994 (Roelke-Parker et al.,
1996) although other CDV outbreaks in
the same seropositive populations had
negligible mortality (Packer et al., 1999).
In pumas, a recent outbreak of feline
leukemia virus has been linked with, but
no causality yet established for, the deaths
of a small number of seropositive Florida
panthers (M. Cunningham, unpubl. data).
The precise role of FIV in regulating
immune response to such opportunistic
infection remains unknown, yet the T-cell
changes observed in FIV-infected lions
and pumas in this study suggest that
further investigation is warranted and that
it would be prudent to discourage the
translocation of seropositive animals into
We thank Catherine Hageman for assis-
tance in western blot assays and Kathleen
Noer for expertise in IFC analysis. We thank
our colleagues W. Johnson, J. Rossio, G. Bar-
Gal, J. Troyer, M. Cunningham, and D. Land.
All tissue samples were collected in full
compliance with specific Federal Fish and
Wildlife permits: Convention of International
Trade in Endangered Species of Wild Flora
and Fauna (CITES) and Endangered and
Threatened Species, Captive Bred issued to
the National Cancer Institute–National Insti-
tutes of Health (S. J. O’Brien, principal officer)
by the US Fish and Wildlife Service of the
Department of the Interior. This publication
has been funded in whole or in part with
federal funds from the National Cancer In-
stitute, National Institutes of Health, under
contract number N01-CO-12400. The content
of this publication does not necessarily reflect
the views or policies of the Department of
Health and Human Services, nor does men-
tion of trade names, commercial products, or
organizations imply endorsement by the US
Government. This research was supported (in
part) by the Intramural Research Program of
the NIH, National Cancer Institute, Center
for Cancer Research. This material is based
upon work supported by the National Science
Foundation under grant 0343960. Collection
of Serengeti lion samples funded in part by
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248JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 2, APRIL 2006