Abstract

Purpose

The purpose of this project was to study in-depth three feline immunodeficiency virus (FIV)-infected lions at the Columbus Zoo. Three uninfected lions served as a control, uninfected group. Detailed immunologic, virologic, biochemical, serologic, neurologic, and histologic evaluations were performed to determine the presence of clinical disease, viral infectivity, and the association of neurologic functional changes with that of immune status and infectivity. Further studies determined gene sequences from virus isolated from FIV-positive lions as a way to evaluate commonalties of the source of the virus.

Study Design

Two adult female (DJ and EL) and one adult male (TT) FIV-infected lions were evaluated (approximate ages of 17, 19, and 15 yr, respectively) at the Columbus Zoo between 1996 and 1998. Three mature, uninfected lions served as a control, uninfected group (two males and one female; approximate ages were 18, 21 and 23 mo). All FIV-infected lions were confirmed positive and control lions confirmed negative by detection of plasma FIV antibody with Western blot analysis using lion-specific antigens. All lions tested negative for feline leukemia virus antigen by ELISA plasma screening.

The specific objectives of the study and associated methodology were to:

1.  Determine the presence of opportunistic infections in relation to the overall health consequences and immunologic deterioration in chronically FIV-infected lions. General health testing consisted of a serum chemistry profile, complete blood count with differential analysis and platelet count, urinalysis, hemostasis screening (OSPT, APTT, fibrinogen, fibrin degradation products), and thoracic radiographs. Diagnostic testing for infectious agents included detection of serum and cerebrospinal antibodies to canine distemper virus, feline infectious peritonitis, Toxoplasma gondii, Neospora caninum and cryptococcosis. Immune status was monitored by lymphocyte subtype analysis and total granulocyte counts at three time points for infected and once for control lions. Lymphocyte subpopulations were enumerated by immunostain and flow cytometry. Results, expressed as numbers of Pan T, CD4 and CD8 lymphocytes, were obtained by multiplying percent positive populations from immunostain with blood count lymphocyte values.

2.  Determine the in vivo neuropathogenicity of chronic FIV infection in lions through neurologic functional testing, cerebrospinal fluid analyses, and magnetic resonance imaging (MRI) scans of the brain. Neurologic function was assessed at two levels: observational changes in behavior and quantitative neurophysiologic changes. Observational changes in behavior and activity were assessed on a monthly basis by trained animal handlers, scored and analyzed for two FIV-infected lions (EL and TT). Selected elapsed time video analysis was reviewed by an independent observer. Frequency of behavior changes were scored on a discrete scale of 0 to 3 (0=normal; 3=agitation, stereotypic movements, or “staring-gazing” activity). Quantitative neurophysiologic changes were analyzed using auditory evoked potential (AEP) and electroencephalography (QEEG) tests. A MRI scan of the brain was performed in one FIV-infected female (EL) on a 0.3 Tesla open field Hitachi AIRIS magnet.

3.  Compare the genetic sequence subtype of the feline immunodeficiency virus (FIV) isolate from FIV-infected lions as compared to FIV-infected snow leopards (2) and a tiger housed at the Columbus Zoo. The DNA was probed from the two snow leopards and the tiger with consensus lion virus primers and ones made specifically from the male FIV-infected lion (TT) virus.

Results

Examination and Observation

Infected lions developed progressive weight loss and mild lymphadenopathy over the study period. All FIV-infected lions were observed to display periodic unusual “star-gazing” activity of variable duration that consisted of motionless staring into the sky with unresponsiveness. Other behavioral changes included dysphagia, abnormal gait and missing benches when jumping from one perch to another. Intermittent periods of lethargy were also seen. A progressive increase in the percentage of abnormal daily observed activity was scored for EL and TT. Both female infected lions were euthanatized due to progressive physical deterioration, while the male lion died of progressive, severe anemia.

Virology

Virus was not amplified in vitro from any of the animals in either primary cells or feline-derived cell lines using standard culture techniques. A 325-base pair sequence was amplified from the pol region of the male infected lion (TT) and sequence analysis indicated that it fell within the "A" clade of lion viruses, yet was distinct and divergent from other isolates that have been analyzed. Primer pairs were designed from this sequence and used in permissive PCR reactions on DNA from the remaining animals. Sequence derived from EL was identical to that amplified from TT using TT primers. No fragments were amplified from the snow leopards or the seropositive tiger using either consensus lion or puma lentivirus primers, or with primers derived from the TT sequence.

Immunology

High titers of serum antibodies ranging from 1:800 to 1:1600 were detected in the three infected lions. All FIV-infected lions demonstrated persistent lymphopenia, and specifically, depletion of CD4 lymphocytes, increase in CD8 counts, and inversion of the CD4: CD8 ratio (Table 1).

Table 1. Comparison of lymphocyte phenotypes in FIV and normal lions (x and range)

Neurodiagnostic Testing

Electrodiagnostic examination: All FIV-infected cats demonstrated abnormalities in AEP and QEEG testing. Prolonged peak onset latencies of waves III and V, and interpeak latencies as compared to the 95% confidence of control cats were seen for the AEP for all FIV-infected cats, indicating altered brain stem pathway function. Infected lions also demonstrated an increase in diffuse slow-wave activity as compared to control lions.

Neuroimaging: The most significant abnormalities associated with the MRI study done on EL were the presence of cerebrocortical atrophy, demonstrated by sulcal widening and secondary ventriculomegaly (hydrocephalus ex vacuo), and symmetric disruption of the deep white matter in the parietal lobe.

Pathology

Complete histopathologic evaluation was performed on all FIV-infected lions with neuropathologic abnormalities detected in all cases. Common to all cases were cerebrocortical atrophy, astrogliosis, and suspected microglial satellitosis. The most severe focal lesions were complete loss of normal white matter of the corpus callosum and adjacent cingulate gyrus with replacement by fibrous connective tissue, presumably of vascular adventitial origin (DJ). Observation of hemosiderin-laden macrophages supports a histopathologic lesion of a cerebral vascular accident in this region. No evidence of opportunistic infections was found in any animal.

Conclusions

The results of this study support the following conclusions:

The general health deterioration observed in these FIV-infected lions was not the result of opportunistic infections.

Phylogenetic analysis of the pol region of the virus from infected lions indicated that this virus was distinct from other lion lentiviruses that have been examined but was most closely related to the "A" clade of lion lentiviruses.

Feline immunodeficiency infection in these zoo lions did result in functional neurologic changes that paralleled progressive decline in CD4 lymphocytes.

1.  Diffuse and focal neuropathologic changes were present in FIV-infected lions that resemble similar changes reported in FIV-infected domestic cats, and HIV-1 infected people.

2.  Overall, this study demonstrates that certain FIV isolates have potential neuropathogenicity with significant health consequences in zoo lions.