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Epizootiology of Feline Leukemia Virus in the Florida Panther


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EPIZOOTIOLOGY OF FELINE LEUKEMIA VIRUS IN THE FLORIDA PANTHER By MARK WILLIAM CUNNINGHAM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Mark William Cunningham

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The role of disease in wildlife conservati on has probably been radically underestimated (Aldo Leopold, 1933).

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ACKNOWLEDGMENTS Most of the information presented in this thesis would not be possible without the expertise of houndsman Roy McBride who began capturing panthers in the 1970s. Also very much appreciated are the efforts of the biologists on the Florida Fish and Wildlife Conservation Commission (FWC) panther capture team including Darrell Land, David Shindle, and Mark Lotz. Veterinarians with FWC who collected samples or data used in this study include Drs. Melody Roelke, Mike Dunbar, Sharon Taylor, Dave Rotstein, and Kristin Mansfield. Researchers with the National Park Service also collected samples and include Deborah Jansen, Steve Schultz, and Dr. Emmett Blankenship. Veterinary pathologists have been especially helpful with this study and include Drs. Scott Terrell, Claus Buergelt, and Bruce Homer. I would especially like to thank my advisor Dr. Donald Forrester for his patience and guidance. I also appreciate the guidance of other committee members including Drs. Julie Levy, Mel Sunquist, and Rick Alleman. I would also like to thank collaborators on this project including Drs. Meredith Brown, Stephen J. OBrien, and Warren Johnson at the National Cancer Institute; and Drs. Kathleen Hayes and Lawrence Mathes at the Ohio State University. I greatly appreciate the advice and support provided by Drs. Scott Citino, Cynda Crawford, and William Hardy, Jr. Finally, I am indebted to Richard Kiltie for assistance with statistical analyses. iv

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This project was fully funded by the Florida Fish and Wildlife Conservation Commission through the Federal Endangered Species Project E-1 and the Florida Panther Restoration and Management Trust Fund. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ...........................................................................................................viii LIST OF FIGURES ...........................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 The Florida Panther...............................................................................................1 Feline Leukemia Virus..........................................................................................4 Objectives...................................................................................................................11 2 MATERIALS AND METHODS...............................................................................13 Study Area and Period................................................................................................13 Florida Panther Capture and Immobilization.............................................................13 Physical Examination..........................................................................................14 Live-capture Sample Collection..........................................................................14 Vaccination and Treatment..................................................................................15 Radio-instrumentation.........................................................................................16 Neonatal Kittens.........................................................................................................16 Necropsy.....................................................................................................................16 Specimen Storage.......................................................................................................17 Age Determination and Genetic Status.......................................................................17 Diagnostics.................................................................................................................17 Enzyme-linked Immunosorbent Assay Antibody...............................................17 Enzyme-linked Immunosorbent Assay Antigen..................................................17 Immunofluorescent Assay and Immunohistochemistry......................................18 Polymerase Chain Reaction, Genetic Sequencing, and Viral Culture.................18 Complete Blood Count and Serum Chemistry....................................................19 Other Diagnostic Testing.....................................................................................19 Statistics......................................................................................................................19 vi

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3 RESULTS...................................................................................................................22 Diagnostic Tests..........................................................................................................22 Enzyme-linked Immunosorbent Assay Antibody...............................................22 Enzyme-linked Immunosorbent Assay Antigen..................................................23 Immunofluorescent Assay and Immunohistochemistry......................................24 Other serology.....................................................................................................24 Clinical Findings.........................................................................................................25 Clinical Pathology...............................................................................................25 Pathology.............................................................................................................25 Gross.............................................................................................................25 Microscopic..................................................................................................26 Opportunistic infections...............................................................................26 Mortality.......................................................................................................26 4 DISCUSSION.............................................................................................................28 Diagnostics.................................................................................................................28 Epizootiology..............................................................................................................30 History of Exposure.............................................................................................30 Prevalence and Distribution................................................................................31 Outcome Following Exposure.............................................................................31 Self-limiting infections.................................................................................32 Persistent infections......................................................................................33 Epizootiology......................................................................................................38 Conclusion..................................................................................................................40 Further Research.........................................................................................................41 APPENDIX A FLORIDA PANTHER/TEXAS PUMAS SAMPLED DURING THE STUDY PERIOD......................................................................................................................42 B CASE REPORTS: ANTIGENEMIC FLORIDA PANTHERS..................................49 FP115...................................................................................................................49 FP109...................................................................................................................51 FP122...................................................................................................................52 FP123...................................................................................................................54 FP132...................................................................................................................55 LIST OF REFERENCES...................................................................................................60 BIOGRAPHICAL SKETCH.............................................................................................70 vii

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LIST OF TABLES Table page A-1 Florida panthers and Texas pumas tested for feline leukemia virus (FeLV) antigen by ELISA 1 July 2002, to 5 June 2005........................................................43 B-1 Selected hematological and serum biochemical values for Florida panthers testing positive for feline leukemia virus (FeLV) antigen by ELISA 1 July 2002 to 5 June 2005..........................................................................................................59 viii

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LIST OF FIGURES Figure page 1-1 Outcome following exposure to feline leukemia virus in domestic cats..................12 2-1 Study area in south Florida, USA.............................................................................21 3-1 Distribution of positive feline leukemia virus positive ELISA antibody optical densities in Florida panthers/Texas pumas by region and year 1990-2005.............23 3-2 Feline leukemia virus (FeLV) ELISA antigen results for panthers 1 yr, not previously FeLV vaccinated, and sampled in South Forida between 1 July 2002 and 5 June 2005........................................................................................................27 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EPIZOOTIOLOGY OF FELINE LEUKEMIA VIRUS IN THE FLORIDA PANTHER By Mark William Cunningham August, 2005 Chair: Donald J. Forrester Major Department: Wildlife Ecology and Conservation Feline leukemia virus (FeLV) has been reported only rarely in non-domestic felids and was not detected in Florida panthers ( Puma concolor coryi ) during almost 20 yr of routine surveillance. The finding of two FeLV antigen-positive panthers during the 2002-2003 capture season led to a prospective and retrospective investigation of the epizootiology of this disease in the population. Archived serum was tested for FeLV antibodies to assess history of exposure. To determine prevalence and distribution, panthers were captured throughout their range and tested for FeLV antigen by ELISA. Positive tests were confirmed by immunofluorescent antibody (IFA) test and viral culture. The outcome following exposure in panthers was inferred from ELISA antigen and antibody, IFA, and PCR results. All infected panthers were monitored by radio-telemetry and necropsied following detection of a mortality signal. Between 1990 and 2005, the prevalence of positive antibody tests increased significantly and were concentrated in the northern portion of panther range. The prevalence of antigenemia (positive ELISA antigen) among panthers and Texas pumas 1 yr of age, not previously x

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vaccinated for FeLV, and sampled between July 2002 and June 2005, was 7% (5 of 71). Antigenemic panthers were captured or recovered in the Okaloacoochee Slough State Forest (OKS) in the northern portion of panther range. All antigenemic panthers were positive by viral culture and three were IFA positive at capture. Clinical signs and clinical pathology at capture ( n = 4) included lymphadenopathy, moderate to severe anemia, lymphopenia, and acute lymphoblastic leukemia. All infected panthers died during the study period; causes of deaths were septicemia ( n = 2), intraspecific aggression ( n = 2), and unknown ( n = 1). Average time from diagnosis to death was 9.25 (SD .3) wk in antigenemic panthers. Following exposure, panthers developed transient, latent, or persistent infections. The high localized prevalence of antigenemic panthers in OKS (45.5%) demonstrates the potential impact of this disease on the population. Management to control the epizootic currently includes vaccination and test-removal. No new cases have been diagnosed since July 2004. xi

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CHAPTER 1 INTRODUCTION The Florida panther ( Puma concolor coryi ) is one of the most endangered mammals in North America, at one time numbering as few as 30 individuals. With protection and management the population has rebounded to almost 100; however, the panther is now threatened by feline leukemia virus (FeLV). Feline leukemia virus infection is a fatal infectious disease, common to domestic cats ( Felis catus ), that is rare in non-domestic felids. Routine FeLV antigen testing in panthers was negative for almost 20 yr until two positive panthers were detected during the 2002-2003 capture season. These findings resulted in a prospective and retrospective investigation into the epizootiology of this disease in the panther population. Information gained from this research will not only be used to help manage the epizootic in this critically endangered population but may also benefit managers of other non-domestic felid populations. Background The Florida Panther The Florida panther is an endangered subspecies of puma whose range was once contiguous with other puma subspecies including the Texas puma ( P concolor stanlyana ). By the early part of the 20 th Century; however, habitat destruction, exploitation, and human population growth had reduced the panther to an isolated remnant population. The panther was eliminated eventually from all previous range with the exception of the relatively inaccessible and, historically, undesirable Big Cypress and Everglades ecosystems of south Florida. Protection of the panther began with state 1

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2 classification as a game animal in 1950 followed by complete state protection in 1958. The panther was listed federally as an endangered species in 1967. Nevertheless, the population dwindled to an estimated 20 to 30 individuals by the early 1970s (Nowak and McBride, 1973). Researchers noticed morphologic differences among panthers from different areas of south Florida. Subsequent genetic analyses revealed two genotypes: 1) original or canonical Florida panthers, concentrated in the Big Cypress ecosystem, and 2) Florida panther/South American puma intercrosses which primarily occupied the Everglades ecosystem (OBrien et al., 1990). The canonical genotype traced its lineage from the original remnant population while the South American puma intercrosses likely resulted from the release of Florida panther/captive puma hybrids into the free-ranging panther population between 1957 and 1967 (Vanas, 1976). Panthers with genetic evidence of South American puma ancestry, although representing a minority, had a greater genetic diversity and fewer congenital anomalies than panthers retaining the canonical genotype (Roelke et al., 1993a). Among canonical panthers, the level of mitochondrial DNA variation, frequency of polymorphic allozyme loci, and average heterozygocity of allozyme loci was lower than any other similarly studied feline except the cheetah ( Acinonyx jubatus ) (OBrien et al., 1990; Newman et al., 1985; Roelke et al., 1993a). The consequences of inbreeding in panthers were believed to have included cryptorchidism (Roelke et al., 1993a; Mansfield and Land, 2002), atrial septal defects (Cunningham et al., 1999), poor seminal traits (Barone et al., 1994), and poor fecundity (Roelke et al., 1993a). Putative impaired immunocompetence was suspected to increase

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3 susceptibility to parasites and infectious diseases including dermatophytosis (Rotstein et al., 1999). Many of these traits are still seen in canonical Florida panthers today. Without intervention the Florida panther was predicted to become extinct within 25 to 40 yr (Seal and Lacy, 1989). However, in 1995 eight female Texas pumas were released into south Florida as part of a genetic restoration program (Seal, 1994). The resultant introgression was designed to restore the genetic diversity to levels comparable to other puma subspecies and to lower the incidence of congenital anomalies in the panther population. As of September 2004, over half of the population had Texas puma genes (D. Land, pers. commun.). The distribution of genotypes was not uniform however, with more canonical panthers present in the northern portion of panther range. Recent microsatellite DNA analyses also provided evidence for a third and more recent introgression. Several captive pumas of unknown western ancestry escaped from the Seminole Indian Reservation (SIR) north of Big Cypress National Preserve (BCNP) between 1996 and 1999. Although most were eventually recaptured, successful breeding with free-ranging panthers apparently occurred, and evidence of this genotype was present in 6-10% of panthers sampled between 2000 and 2004 (D. Land, pers. commun.). This genotype was concentrated also in the northern portion of panther range. The prevalence of congenital anomalies among intergrades was reduced greatly and was limited to the occasional kinked tail or cowlick. As a result of the genetic introgressions, both deliberate and unintentional, and other management measures, the panther population had rebounded to a minimum of 87 by 2003 (McBride, 2003). However, this increase in density may have resulted in an increased risk of infectious

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4 disease transmission and expansion of the wildland-urban interface. These factors may have set the stage for the current FeLV epizootic. Feline Leukemia Virus Feline leukemia virus is a Gammaretrovirus in the family Retroviridae. Following penetration of the host cell by the viral RNA, reverse transcriptase transcribes viral RNA into double-stranded DNA which is then incorporated into the host genome. Incorporated viral DNA, known as provirus, codes for viral proteins and serves as a template for the production of viral RNA. There are numerous strains of FeLV and few isolates in nature are identical (Hoover and Mullins, 1991). Feline leukemia virus is classified into subgroups A, B, and C based on envelope antigens (Jarrett et al., 1973; Sarma and Log; 1973). All FeLV-infected cats carry subgroup A (Jarrett et al., 1978), which is the least pathogenic and only transmissible form. Subgroup C results from mutation of subgroup A while subgroup B arises from recombination between subgroup A and endogenous retroviral DNA (enFeLV) (reviewed by Miyazawa, 2002). EnFeLV are non-coding, non-immunogenic sequences (Mandel et al., 1979; Rigby et al., 1992) that became incorporated in the domestic cat genome early in their phylogenetic history. Most non-domestic felids, including Florida panthers, do not have enFeLV. The domestic cat is the definitive host for FeLV and the virus has a worldwide distribution. Although several non-felid cell lines have shown in vitro susceptibility (Nakata et al., 2003) infection has not been described in non-felid species. The worldwide prevalence of FeLV in healthy domestic cat populations ranges from 1-8% (Levy, 1999) with prevalences over 30% in some closed populations (Grant et al., 1980; Gertsmann, 1985). There is evidence that the overall prevalence of FeLV in domestic cats is decreasing, possibly due to vaccination and other control measures (Levy and Crawford,

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5 2005). In Florida, the prevalence among feral cats is less than 4% (Lee et al., 2002). Infection is more prevalent among male cats, mixed breed cats, and cats between 1 and 7 yr of age (Levy, 2005). The highest infection rate occurred in cats less than 2 yr of age (Levy, 1999). In contrast, the prevalence of FeLV antibodies, indicating exposure, continues to increase with age (Rogerson et al., 1975). Feline leukemia virus is an enveloped virus and is therefore quite fragile. The virus immediately begins losing viability outside of the host and, on dry surfaces, is completely inactivated between two and three hr (Francis et al., 1979). Therefore transmission is primarily by direct contact. The virus is shed in highest concentrations in the saliva (Francis et al., 1977), and horizontal transmission occurs primarily via the oronasal route and by bite wounds. Prolonged contact is generally necessary for effective transmission (Hardy et al., 1973). Transplacental and transmammary transmission of the virus are also important (Hardy et al., 1976). Following exposure most domestic cats will eventually clear the virus while approximately one-third will become persistently infected and eventually succumb to FeLV related diseases. However, there is a dynamic relationship between the host and virus, and progression of disease depends on a number of factors. Outcome following exposure depends on host age (Hoover et al., 1976), genetics (Hoover and Mullins, 1991), and immunocompetence (Hoover et al., 1980), as well as route of exposure, virus burden, and strain of virus (Rojko and Kociba, 1991; Hoover and Mullins, 1991). The progression of infection can be predicted by provirus burden using quantitative polymerase chain reaction (PCR) (Hofmann-Lehmann et al., 2001). Cats clearing infection early have no or low provirus burdens, those latently infected retain moderate

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6 levels of provirus, while those becoming persistently infected have high provirus burdens that peak at about 4 wk post-exposure (Hofmann-Lehmann et al., 2001). These researchers also demonstrated an inverse correlation between ELISA antibodies and provirus load beginning approximately 3 wk post-exposure. Cats that resisted persistent infection had a more pronounced humoral response and lower provirus burdens than cats that progressed to persistent infections. Cell-mediated immunity is important also in the early immune response to FeLV infection (Flynn et al., 2002). Regardless of the outcome, the course of infection is established usually by 8 wk post-exposure (Torres et al., 2005). Outcome following exposure in domestic cats is summarized in Fig. 1-1. Following exposure the virus replicates in local lymphoid tissues. Approximately 40% of cats mount an effective immune response and clear the virus before further progression (Hoover and Mullins, 1991). These cats remain antigen and provirus negative throughout their lives (Torres et al., 2005). If the infection progresses, however, viral replication within a small number of circulating leukocytes will lead to infection of lymphoid organs including the thymus, spleen, and lymph nodes (Rojko et al., 1979). Cats at this stage may be transiently antigenemic and may even be briefly infectious. Clinical signs during this primary viremia may include fever, lethargy, leukopenia, anemia, and lymphadenopathy (Pedersen et al., 1990; Levy, 1999). However, approximately 50% of cats reaching this stage are still able to mount an effective immune response and clear the infection (Hoover and Mullins, 1991). Failure of viral containment will lead to infection of the bone marrow, salivary glands, and other tissues between 3 and 13 wk. Nevertheless, an adequate immune response early in this process may rescue the cat from persistent infection. These cats will retain provirus in peripheral and marrow

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7 leukocytes for variable periods and are considered latently infected. Latently infected cats do not shed virus and are not infective to other cats. Reactivation of latent infections following stress is possible but becomes less likely 1 yr post-infection (Pedersen et al., 1984). Generally, cats recovering from transient or latent infections are immune to re-infection. Progression to persistent infection occurs in approximately 35% of exposed cats and is characterized by infection of the bone marrow and the development of cytosuppressive and cytoproliferative diseases. Severity and type of disease in persistently infected cats depends on host age (Hoover et al., 1976), concurrent feline immunodeficiency virus (FIV) infection, and virus subgroup and strain. Following establishment of a persistent infection, a period of dormancy ensues lasting weeks to years during which few if any clinical signs are apparent. Eventually, persistent infections result in any of three clinical syndromes: immunosuppression, anemia, and/or neoplasia. Immunosuppression is believed to result in opportunistic infections. Co-infections were the most frequent finding in FeLV infected cats examined at North American veterinary schools (Levy, 1999). Anemia, whether primary or secondary, is the next most common clinical finding in FeLV infected cats. Anemias are most commonly non-regenerative and include pure red cell aplasia, red blood cell macrocytosis, erythemic myelosis, bone marrow infiltration, and anemia of chronic disease (Hardy, 1980a). Finally, hematopoietic neoplasms may also result from FeLV infection. Lymphoma is the most common FeLV-related neoplastic disease; leukemias, myeloproliferative diseases, and fibrosarcomas are also common (Hardy, 1980a). Mortality among persistently

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8 infected cats is approximately 5-fold that of uninfected cats and 83% die within 3.5 yr (McClelland et al., 1980). Co-infections of FIV and FeLV are believed to work synergistically to result in more severe disease (Grindem et al., 1989; Pedersen et al., 1990; Hofmann-Lehmann et al., 1997). Beebe et al. (1994) suggested that immunosuppression caused by pre-existing FeLV infection affected disease development upon subsequent FIV infection. Feline immunodeficiency virus infected cats experimentally infected with FeLV had more severe disease with a more rapid onset than cats infected with either virus alone. Further, CD4+ T-lymphocytes were much more depressed in co-infected cats than cats infected with either virus alone (Hoffmann-Lehmann et al., 1995). It is unknown whether the order of infection (FeLV or FIV first followed by the other) is important in the clinical outcome (Hofmann-Lehmann et al., 1997). Finally, virus/virus interactions such as the formation of FeLV/FIV pseudotypes does not appear to be a mechanism of disease potentiation (Beebe et al., 1994). The outcome following introduction of FeLV into nave domestic cat populations depends on population size, density, dispersal patterns, and spatial and social structure (Fromont et al., 1998a,b; Fromont et al., 2003). Based on computer models, FeLV becomes established in large natural domestic cat populations at a prevalence of between 0.8% and 12.4% depending on the parameters used (Fromont et al., 1998a,b) and reduces population size by 3% (Courchamp et al., 1997) to 7% (Fromont et al., 1997). Inclusion of FIV in Courchamps et al. (1997) models more than doubled the population impact of FeLV. Fromont et al. (1998a) also predicted that FeLV fails to become established in

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9 small isolated populations numbering <100 individuals although extinction of the virus may take several years. Feline leukemia virus can be diagnosed and staged using a variety of techniques. The enzyme-linked immunosorbent assay (ELISA) antigen test is the most common screening method. The ELISA detects soluble p27 antigen in blood (Lutz et al., 1980a) usually within 3 wk post-infection (Hofmann-Lehmann et al., 2001). Positive test results may indicate transient or persistent infection and are an indicator of viremia. Confirmation of positives is accomplished by immunofluorescent assay (IFA), which detects p27 antigen within neutrophils and platelets of blood smears (Hardy et al., 1973). A positive IFA test indicates infection of the bone marrow and usually indicates persistent infection. Viral culture is highly specific and may be used to detect transient, latent, or persistent infections and to identify subgroup. Polymerase chain reaction is a highly sensitive and specific technique that has been used to detect integrated provirus or free FeLV in formalin-fixed tissues, fresh tissues, bone marrow, and blood. Most transient and persistent infections are detectable by PCR 1 wk post-infection and all are detectable by 2 wk (Hofmann-Lehmann et al., 2001). Detection of FeLV antibodies helps stage the disease, especially identifying previous transient infections, but has little importance in diagnosis. Feline leukemia virus ELISA antibodies are most frequently found in those groups clearing the infection (Lutz et al., 1980b). Finally, sequencing of virus is used to identify strain and subgroup. Expected test results during various stages of FeLV infection are summarized in Fig. 1-1. Infection of non-domestic felids by FIV, also a retrovirus, is relatively common and usually does not result in clinical signs. Approximately 28% of Florida panthers carry the

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10 puma lentivirus strain of FIV (Olmstead et al., 1992) and pathology has not been observed. In contrast to FIV, FeLV infections in non-domestic felids are quite rare. Feline leukemia virus infection has been documented in a handful of captive non-domestic felids including a bobcat ( Lynx rufus ) (Sleeman et al., 2001), puma (Meric, 1984), clouded leopard ( Neofelis nebulosa ) (Citino, 1986), and several cheetahs ( A jubatus ) (Briggs and Ott, 1986; Marker et al., 2003). Feline leukemia virus has also been isolated from a leopard cat ( F bengalensis ) cell line (Rasheed and Gardner, 1981). In all cases, the source of infection was believed to be infected domestic cats. Despite extensive testing for FeLV in free-ranging felid populations (Rasheed and Gardner, 1981; Mochizuki et al., 1990; Roelke et al., 1993b; Paul-Murphy et al., 1994; Hofmann-Lehmann et al., 1996; Miyazawa et al., 1997; Osofsky et al., 1996; Biek et al., 2002; Munson et al., 2004; Riley et al., 2004; Ryser-Degioris et al., 2005) published reports of FeLV infection have been limited to a puma ( P concolor ) in California (Jessup et al., 1993) and a sand cat ( F margarita ) in Saudi Arabia (Ostrowski et al., 2003). Approximately 10 to 24% of European wildcats ( F sylvestris sylvestris ) were also FeLV positive (Daniels et al., 1999; Fromont et al., 2000), although interbreeding with domestic cats occurs frequently in this subspecies (Daniels et al., 1998). There have been reports of positive FeLV test results in free-ranging non-domestic felids that were not confirmed with additional tests. Rickard and Foreyt (1992) detected FeLV antigen in 2 of 2 free-ranging pumas found dead in Washington but virus isolation was not attempted. Schmitt et al. (2003) diagnosed FeLV infection by IFA in 11 of 16 (69%) captive and free-ranging felids from Brazil, a biologically inconsistent percentage, but did not confirm the results by ELISA antigen or viral culture.

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11 Testing for FeLV antibodies has been performed only rarely on samples collected from non-domestic felids. Feline leukemia virus antibodies were found in a transiently infected captive clouded leopard (Citino, 1986) and two captive Siberian tigers ( Panthera tigris altaica ) (Meric, 1984). Most infections in non-domestic felids were self-limiting. In a survey of North American zoos, 7 of 11 (64%) non-domestic felids that originally tested FeLV-positive, were negative when retested. The remaining four were not retested and did not go on to develop clinical signs of FeLV. Clinical signs in non-domestic felids with self-limiting infections were minimal and included lethargy, peripheral lymphadenopathy, and dehydration. Terminal infections were seen in a free-ranging and captive puma, a bobcat, and a cheetah. Clinical pathology and necropsy findings included anemia, lymphopenia, other cytopenias, septicemia, lymphadenopathy, opportunistic infections, and lymphoma (Meric, 1984; Jessup et al., 1993; Sleeman et al., 2001; Marker et al., 2003). In Florida panthers, routine FeLV ELISA antigen testing was negative since testing began in 1978 through late 2002 (Roelke et al., 1993b; Florida Fish and Wildlife Conservation Commission, unpubl. data); however, during the 2002-2003 capture season, two panthers tested antigen-positive. These findings launched the investigation detailed in this report. Objectives The objectives of this study were to determine for FeLV in Florida panthers 1) the history of exposure, 2) the prevalence and geographic distribution, 3) the outcome following exposure, 4) the clinical signs, clinical pathology, and pathological changes associated with infection, and 5) risk factors for infection.

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12 Exposure DIrect contact Fighting Nursing Abortive Infection Antibody + Antigen IFA Provirus No clinical disease Most immune to reinfection Latent Infection Antibody + Antigen IFA Provirus ++ May revert to persistent infection but unlikely after >1 yr Immune to reinfection Transient Infection Antibody + Antigen IFA Briefly provirus + No clinical disease Immune to reinfection Viremia Antibody +/Antigen + IFA Provirus + to +++ 3 wks ~35% of those exposed will remain persistently infected ~65% eventually clear virus Elimination of Latent Infection Antibody + Antigen IFA Provirus Immune to reinfection 5% Days 3-13 wks Viremia Antibody +/Antigen + IFA + Provirus ++ to +++ Infectious Persistent Infection Antibody +/Antigen + IFA + Proivirus +++ Infectious, most die <3 yrs Life Adapted from: Hartmann (2005), Hoover and Mullins (1991), and Torres et al. (2005). 33% 33% 33% 5% Atypical Infection Antibody +/Antigen +/IFA +/Provirus +/10% INFECTION CLEARED ACTIVE INFECTION Figure 1-1. Outcome following exposure to feline leukemia virus in domestic cats.

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CHAPTER 2 MATERIALS AND METHODS Study Area and Period Florida panthers were sampled in southern peninsular Florida (south of 28 N) primarily in the Big Cypress and Everglades ecosystems. For ELISA antibody comparisons, capture/sampling locations were divided into north and south of I-75 (approximately 28.05 N) (Fig. 2-1). The prospective study period was 1 July 2002 to 5 June 2005. Archived tissues collected between 1990 and 30 June 2002 were retrospectively evaluated. For analysis of ELISA antibody prevalence, the study period was divided into before (1990-1995) and after (1996-2005) genetic restoration. Previously published and unpublished FeLV ELISA antigen test results from 1983 to 30 June 2002 are included in this report (Roelke, 1990; Roelke et al., 1993b; Dunbar, 1994; FWC, unpubl. data). Florida Panther Capture and Immobilization Free-ranging Florida panthers and translocated Texas cougars were captured using trained hounds. Panthers either bayed on the ground or were treed, and then were darted with a 3 ml compressed-air dart fired from a CO 2 -powered rifle. Since 2002, immobilization drugs included various combinations of ketamine HCl (Congaree Veterinary Pharmacy, Cayce, South Carolina, USA), medetomidine (Domitor, Pfizer Animal Health, Exton, Pennsylvania, USA), tiletamine HCl/zolazepam HCl (Telazol, Fort Dodge Animal Health [FDAH], Fort Dodge, Iowa, USA), midazolam HCl (Abbott Laboratories, North Chicago, Illinois, USA), and xylazine HCl (Congaree Veterinary 13

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14 Pharmacy, USA) (Shindle et al., 2003; Shindle et al., 2004). Following immobilization, treed panthers were caught with a net and, in some cases, a crash bag (McCown et al., 1990). Propofol (PropoFlo TM Abbott Laboratories, North Chicago, Illinois, USA) was administered intravenously (IV) either as a bolus or continuous drip to maintain anesthesia. Butorphanol tartrate (0.1-0.4 mg/kg, FDAH) or midazolam HCl (0.03 mg/kg) was administered intramuscularly (IM) to smooth recovery in some panthers. Panthers were left to recover in a shaded area away from water. Xylazine HCl and medetomidine HCl were reversed with yohimbine HCl (Yobine, Lloyd, Inc., Shenandoah, Iowa, USA) and atipamezol HCl (Antisedan, Pfizer Animal Health, Exton, Pennsylvania, USA), respectively, at to their recommended dosages. Physical Examination Vital signs (temperature, heart rate, respiration rate, and capillary refill time) and depth of anesthesia were monitored and recorded. A sterile petrolatum ophthalmic ointment was applied to the eyes for lubrication. All animals underwent a physical examination to assess general health and physical condition. For each panther handled, the skin over the medial saphenous vein was clipped, prepped, and an IV catheter aseptically placed. Sterile isotonic fluids were administered either subcutaneously (SQ) or IV. Panthers were implanted with a SQ transponder identification chip (Trovan, Douglas, United Kingdom), ear-tattooed, measured, and weighed. Live-capture Sample Collection Approximately 70-140 ml of blood (depending on body weight) were collected from the medial saphenous or cephalic veins using a butterfly catheter (19 or 21 gauge), luer adapter/hub, and Vacutainer tubes (Becton Dickinson, Franklin Lakes, New Jersey, USA) (approximately 50 ml in serum separator, 40 ml in EDTA, 9 ml in Na Heparin, and

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15 9 ml in ACD tubes). From uncollared panthers, eight skin biopsies (4 mm) were collected aseptically from the medial aspect of the hindlimbs and saved in biopsy transport media. Defects were closed with surgical glue. Hair clipped from blood collection and biopsy sites and pulled hair were saved in sample collection bags; clipped hair was saved also from the ventral abdomen. Other samples such as bacterial cultures, skin scrapings, and diagnostic biopsies were taken if indicated. Between November 2002 and April 2004, blood smears were made from EDTA whole blood on glass slides approximately 6 to 24 hr after collection. Beginning May 2004, blood smears were made in the field from fresh whole blood. Vaccination and Treatment Panthers >4 mo old were vaccinated SQ against feline viral rhinotracheitis (FVR), feline calicivirus (FCV), feline panleukopenia virus (FPV) (Fel-O-Vax PCT [FDAH], 1 ml, lower left leg), and rabies (Rabvac TM 3 [FDAH], 1 ml, lower right leg). Beginning June 2003, captive and free-ranging panthers were also vaccinated against feline leukemia virus (FeLV, Fel-O-Vax Lv-K [FDAH] or Fevaxyn FeLV, Schering-Plough Animal Health Corporation, Omaha, Nebraska, USA, 2 ml, lower left leg). Some panthers were given a FeLV booster (2 ml) IM remotely by darting 3-16 wk post initial inoculation. Captured panthers were dewormed with ivermectin (0.1 mg/kg, Ivomec, Merial Limited, Iselin, New Jersey, USA) and praziquantel (3.75 mg/kg, CestaJect TM Phoenix Pharmaceutical, Inc., St. Joseph, Missouri, USA) administered SQ in the lateral aspect of thigh. Penicillin G procaine/benzathine (USVet, Hanford Pharmaceuticals, Syracuse, New York, USA) was administered IM at 22,000 to 44,000 U/kg.

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16 Radio-instrumentation Captured adult and juvenile panthers were fitted with a VHF or VHF/GPS radio-collar and monitored three times weekly as described by Shindle et al. (2004). If a mortality signal was detected the carcass was recovered the same day for necropsy. Neonatal Kittens Neonatal kittens were handled according to Land et al. (1998) and marked with a SQ transponder identification chip. Pyrantel pamoate (22 mg/kg, Anthelban V, Phoenix Pharmaceutical, Inc., St. Joseph, Missouri, USA) was administered orally. Blood was collected from the jugular vein. Necropsy All FeLV-positive Florida panthers and/or those found dead due to infectious disease or unknown causes were completely necropsied by board-certified pathologists at the University of Florida (Veterinary Medical Teaching Hospital, Gainesville, Florida, USA) or Disneys Animal Kingdom (Celebration, Florida, USA). One severely autolyzed FeLV-positive panther (FP109) and all panthers dying of known trauma were necropsied by the FWC veterinarian at the Wildlife Research Laboratory (FWC, Gainesville, Florida, USA). When carcass condition allowed, tissue samples were collected at necropsy from all major organs. Fluids collected included heart blood, venous blood, thoracic blood, aqueous humor, and urine. Blood samples were centrifuged at 2000 rpm for 10 minutes and the supernatant decanted. Representative tissues from fresh (unfrozen) and some previously frozen panthers were placed in 10% neutral buffered formalin. Fixed tissues were embedded in paraffin, sectioned at 5 to 6 m and stained with hematoxylin and eosin.

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17 Specimen Storage All tissues from live-captured and necropsied panthers not immediately analyzed were archived at to C. Age Determination and Genetic Status Panther ages were either known (handled as kittens) or were estimated from tooth wear. Panthers were classified as neonates (<8 wk-old), dependents (8 wk to <1 yr), subadults (1 to <2.5 yr), adults (2.5 to <10 yr), and older adults (10 yr). Panthers were grouped by genotype (canonical Florida panther, Texas puma, Texas puma/Florida panther intergrade, Texas puma/Everglades/Florida panther intergrade, SIR/Florida panther intergrade, and Everglades/Florida panther intergrade) (W. Johnson, unpubl. data). Diagnostics Enzyme-linked Immunosorbent Assay Antibody Antibodies to FeLV were detected at Hansen Veterinary Immunology (Dixon, California, USA) according to techniques described by Lutz et al. (1980b). Optical densities (OD) of less than 0.25 were considered negative, 0.25 to <0.35 were low positive, 0.35 to <0.5 were medium positive, and those 0.500 were high positive. For statistical analysis any OD 0.25 was considered positive. Enzyme-linked Immunosorbent Assay Antigen Serum for ELISA antigen testing (ViraCHEK FeLV, Synbiotics Animal Health, San Diego, California, USA) was shipped overnight to the New York State Diagnostic Laboratory (Cornell University, Ithaca, New York, USA). Adsorbing reagents were used to remove heterophile antibody. Fluids collected from live-captured and necropsied panthers were tested for FeLV antigen with a rapid immunochromatic assay (SNAP

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18 Combo, IDEXX Laboratories, Westbrook, Maine, USA). Beginning November 2003, EDTA whole blood from captured panthers was tested in the field using the SNAP combo. The SNAP Combo was also used to test fluids collected from necropsied panthers. Fluids included blood collected from the thoracic cavity, heart chambers, vessels, and marrow cavity, and aqueous humor. Immunofluorescent Assay and Immunohistochemistry Panthers testing positive by ELISA antigen were also tested by IFA. Immunofluorescent assays were performed on EDTA or fresh whole blood smears using techniques described by Hardy et al. (1973) at the National Veterinary Laboratory (Franklin Lakes, New Jersey, USA). Immunohistochemistry to identify p27 antigen was performed on formalin-fixed paraffin-embedded tissues at the Diagnostic Center for Population and Animal Health (Michigan State University, Lansing, Michigan, USA) using a labeled streptavidin-biotin peroxidase detection system on an automated stainer (Ramos-Vara et al., 2002). Polymerase Chain Reaction, Genetic Sequencing, and Viral Culture Polymerase chain reaction and subsequent genetic sequencing was performed at the Laboratory for Genomic Diversity (National Cancer Institute, Frederick, Maryland, USA) on tissues collected from panthers at capture and necropsy. Viral culture was performed at the Center for Retrovirus Research (The Ohio State University, Columbus, Ohio, USA). Materials and methods used, and complete results for PCR and genetic sequencing (M. Brown, unpubl. data) and viral culture (K. Hayes, unpubl. data) will be presented in separate reports.

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19 Complete Blood Count and Serum Chemistry Complete blood counts (CBC) and serum biochemical parameters were determined by Antech Diagnostics (Smyrna, Georgia, USA). Blood smears were examined at the Veterinary Medical Teaching Hospital (University of Florida, College of Veterinary Medicine, Gainesville, Florida, USA) for hemoparasites, white blood cell differential counts, and red blood cell morphology. Other Diagnostic Testing Necropsied panthers were tested for rabies by IFA at the Jacksonville Central Laboratory (Jacksonville, Florida, USA). Viral isolation and real-time and conventional PCR for canine distemper virus (CDV), pseudorabies virus, flaviviruses, and alphaviruses were performed at the Southeastern Cooperative Wildlife Disease Study (Athens, Georgia, USA) on brain, heart, and other tissues collected from panthers dying of unknown causes. Other serological tests included Western blot for FIV and kinetics-based enzyme-linked immunosorbent assay (KELA) for feline coronavirus antibodies (FCV) (New York State Diagnostic Laboratory). Polymerase chain reaction for Mycoplasma haemofelis and M haemominutum was performed on EDTA whole blood from FeLV positive panthers at the University of Illinois (College of Veterinary Medicine, Urbana, Illinois, USA) and Cornell University (Ithaca, New York, USA). Statistics Prevalence was calculated as the percentage of panthers/pumas positive for FeLV antibodies by ELISA (OD >0.251). Raw prevalence estimates were examined for each potential categorical predictor (age classs, genotype, FIV status, location, time period, and gender). Logistic regression using Egret software ( Cytel Software Corporation,

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20 Cambridge, Massachusetts, USA) was performed to investigate ELISA antibody status as a binary response variable. Odd ratios and their 95% confidence limits were calculated for each state of the categorical predictors in comparison to an arbitrary reference state. Significance of difference from 1.0 was determined for the odd ratios by the Wald test. To account for correlation among replicate outcomes from individuals with multiple test results, panther identification was modeled as a random effect within the logistic regression model. Significance of the random effect was evaluated by a likelihood ratio test. Test results were considered significant at P <0.05. The two significant predictors emerging from univariate analyses (location and time period) were included in a multiple predictor logistic regression analysis and their interactions examined.

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21 Figure 2-1. Study area in south Florida, USA.

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CHAPTER 3 RESULTS Diagnostic Tests Enzyme-linked Immunosorbent Assay Antibody ELISA antibody ODs were determined for samples collected from 128 Florida panthers/Texas pumas on 257 occasions between 2 January 1990 and 29 March 2005. Eighteen (7%) samples from 17 individuals were positive (1 high OD, 3 medium OD, 14 low OD). The prevalence of positive antibody ODs was significantly greater in the period 1996-2005 compared to 1990-1995 ( P = 0.032). The prevalence of positive antibody ODs was significantly greater among panthers sampled north of I-75 compared to south ( P = 0.014). No positive ODs were found in the southern portion of panther range (south of US41). The odds of having a positive antibody OD were not affected by age, gender, genotype, or FIV status. Of panthers sampled on multiple occasions, six had low or medium positive ODs at their initial sampling but seroconverted to negative status when re-sampled 10 mo to 3 yr later. 22

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23 90919293949596979899000102030405 SouthCentralNCNorth 00.10.20.30.40.50.60.70.80.91 Figure 3-1. Distribution of positive feline leukemia virus positive ELISA antibody optical densities in Florida panthers/Texas pumas by region and year 1990-2005. North refers to lands north of CR846, NC refers to lands between CR846 and I-75, Central refers to lands between I-75 and US41, and South refers to lands south of US41. Enzyme-linked Immunosorbent Assay Antigen Prior to the study period, all ( n = 143 sampled on 322 occasions) Florida panthers and Texas pumas sampled were negative for p27 antigen by ELISA based on review of published and unpublished data and retrospective testing. During the study period (1 July 2002 to 5 June 2005), 91 panthers/Texas pumas were tested on 113 occasions for FeLV antigen by ELISA. Fifty-five panthers or pumas were sampled on 66 occasions at capture, 40 were sampled at necropsy, 10 were sampled at both capture and necropsy, and seven were tested as neonatal kittens. Panther number, age, gender, FIV status, and results of FeLV diagnostic tests are presented in Table A-1.

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24 The prevalence of antigenemia (positive ELISA antigen) among panthers and Texas pumas 1 yr of age, not previously vaccinated for FeLV, and sampled during the study period, was 7% (5 of 71). All antigenemic panthers were captured in Okaloacoochee Slough (OKS). The prevalence of antigenemia in OKS (NC region, Fig. 3-2) was 45.5% (5 of 11). Antigenemia was only detected in adult panthers (3 males, 2 females). The average age of antigenemic panthers was 4.85 yr (standard deviation [SD].5) and ranged from 2.25 to 11 yr. Genotypes included canonical Florida panthers ( n = 3), Texas puma/Florida panther intergrade ( n = 1), and SIR captive/Florida panther intergrade ( n = 1). Case histories of antigenemic panthers are presented in Appendix B. Feline leukemia virus antigen was detected by SNAP test in all fluids tested in those viremic panthers suitable for testing at necropsy. Fluids testing positive included thoracic blood (FP115, 122, 123, 132), splenic blood (FP115), venous blood (FP132), and aqueous humor (FP115, 122, 123, 132). Immunofluorescent Assay and Immunohistochemistry Three (FP122, 123, 132) of the 5 (60%) panthers positive for FeLV antigen by ELISA were also IFA positive. Results for two viremic panthers (FP109, 115) were inconclusive. Spleen and lymph node from 2 of 2 (100%) viremic panthers (FP115, 132) were positive for p27 antigen by IHC. Other serology During the study period, 37.5% of panthers/Texas pumas tested were positive for FIV antibodies by Western blot. Three of five (60%) FeLV antigen-positive panthers also tested positive for the puma lentivirus strain of FIV (J. Troyer, unpubl. data). Serology for FCV was negative for all panthers sampled during the study period ( n = 64).

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25 Clinical Findings Clinical signs observed at capture in four antigenemic panthers included a peripheral lymphadenopathy ( n = 2, 50%) and muscle wasting ( n = 1, 25%). Clinical Pathology Complete blood counts were performed on four antigenemic panthers sampled while living. Significant findings included a mild to moderate non-regenerative anemia ( n = 3 [75%]), lymphopenia ( n = 3), low hemoglobin ( n = 3), monocytosis ( n = 1 [25%]), and elevated nucleated red blood cell count ( n = 1). Large immature mononuclear cells with prominent nucleoli, consistent with acute lymphoblastic leukemia, were seen in two panthers (FP122, 123, 50%). The mean hematocrit of antigenemic panthers was 29.3% (SD 7.9, range 22.5-42.5%), hemoglobin 9.3 g/dL (SD.4, range 7.2-13.2 g/dL), red blood cell count 6.2 x 10 6 /l (SD.78 x 10 6 /L, range 4.2-8.75 x 10 6 /l), and lymphocyte count 1165/l (SD.9/l, range 490-2250/l). Serum biochemical values in antigenemic panthers were unremarkable. Clinical pathology of antigenemic panthers and normal values for panthers are summarized in Table B-1. Pathology Gross Three antigenemic panthers (FP115, 122, 132) were suitable for complete necropsy based on carcass condition. Completely necropsied panthers had evidence of anemia (pale mucus membranes and skeletal muscle, n = 2 [66.7%]), moderate to severe dehydration ( n = 2), lymphadenopathy ( n = 2), septicemia ( n = 2), bronchointerstitial pneumonia ( n = 2), abscesses ( n = 1, [33.3%]), and puncture wounds ( n = 1). Lacerations and puncture wounds associated with intraspecific aggression (ISA) were seen in the two autolyzed/decomposed carcasses (FP109, 123).

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26 Microscopic Microscopic examination was performed on three panthers (FP115, 122, 132). Sections of bone marrow from two (66.7%) panthers were hypercellular with approximately 90 to 100% of the marrow space occupied by hematopoietic cells. Megakaryocytes were present in normal to moderately increased numbers. No marrow evidence of acute lymphoblastic leukemia was seen. Microscopic changes consistent with septicemia were seen in most tissues in FP115 and 132. See Appendix B for complete histological results. Opportunistic infections Aerobic culture of multiple tissues from FP115 and 132 resulted in heavy growth of Escherichia coli and -hemolytic Streptococcus sp., respectively. Rabies IFA and viral isolation and PCR for canine distemper virus, pseudorabies virus, flaviviruses, and alphaviruses were negative in FP122. Two of four (50%) ELISA antigen-positive panthers (FP109, 115) were PCR positive for M hemominutum ; FP115 was also positive for M haemofelis (J. Messick, unpubl. data). Organisms were not seen on blood smears made from EDTA whole blood. Rare Cytauxzoon felis organisms were seen on blood smears from FP109; blood smears from FP115, 122, and 123 were negative. Mortality Suspected causes of death for the five antigenemic panthers included septicemia ( n = 2), intraspecific aggression ( n = 2), and anemia/dehydration ( n = 1). Time from diagnosis to death averaged 9.25 (SD.3, range 2-24.6) wk in panthers antigenemic at capture (FP109, 115, 122, 123). Time from diagnosis to death in the two panthers believed to have died due to FeLV-related diseases was 2 (FP122) and 24.6 wk (FP115).

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27 Time from exposure to death for one panther (FP132) dying of FeLV-related disease was 18.4 wk. Figure 3-2. Feline leukemia virus (FeLV) ELISA antigen results for panthers 1 yr, not previously FeLV vaccinated, and sampled in South Forida (south of Caloosahatchee River) between 1 July 2002 and 5 June 2005.

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CHAPTER 4 DISCUSSION Diagnostics Diagnostic tests validated for domestic animals but used on wildlife must be interpreted with caution (Hietala and Gardner, 1999). Nevertheless, the test results in this study were biologically consistent and appeared to be appropriate and suitable for use in panthers. The ELISA antibody test detects exposure to FeLV and is considered more sensitive but less specific than Western blot analysis. ELISA antibody testing has only been used rarely in non-domestic felids. Ryser-Degiorgis et al. (2005) found serum from 58 of 102 (58%) Eurasian lynx ( L lynx ) to be FeLV positive by ELISA antibody but negative by Western blot. The authors speculated that cross-reactions with E coli antigen (test preparation) or antibodies to murine leukemia viruses may have been responsible for the false-positive results. Our positive ELISA antibody tests were not confirmed by Western blot. Further, antibody ODs in panther serum were tested incrementally. Incremental testing of serum for ELISA antibodies may lead to inconsistent results due to between-batch variation. Additionally, degradation of antibodies in stored serum may have resulted in the apparent increase in positive ODs in recently collected samples. However, FeLV antibodies are stable when frozen in serum (S. Hansen, pers. commun.). Additionally, ELISA antibody results in Florida panthers were consistent biologically with other test results and observations. Panthers seroconverted following vaccination (data not shown) and positive ODs were geographically and temporally clustered. 28

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29 ELISA antigen tests detect the FeLV p27 protein and therefore should be suitable for use in exotic species. Nevertheless, false-positives have occurred in some tests that used murine-derived reagents in domestic and non-domestic cats that had naturally occurring anti-murine antibodies (Lopez and Jacobson, 1989). False positive results were reported in one Florida panther tested in 1987 (Lopez, 1988). In this case anti-mouse antibodies were believed to have resulted from vaccination with a rabies vaccine of mouse brain origin. Changes in test procedures and reagents effectively eliminated this problem by the early 1990s (Jacobson and Lopez, 1991). False positives may occur also due to insufficient washing of vessels in micro-well systems (Jarrett et al., 1982), a problem not encountered when using rapid immunoassay test kits. The effectiveness of using body fluids from known infected panthers for detection of p27 antigen was evaluated. Hemolyzed thoracic, heart, and venous blood; bone marrow; and aqueous humor from infected panthers consistently tested positive by rapid immunoassay (SNAP Combo), even on severely autolyzed specimens. The p27 antigen is only 27,000 daltons and is thus small enough to cross into the aqueous humor in healthy felids (K. Gellatt, pers. commun.). Thus aqueous humor, and the other fluids described above, may be useful for FeLV monitoring not only in panthers but in other populations of pumas. Immunofluourescent assay and IHC detect p27 antigen in platelets and neutrophils of blood smears and paraffin-embedded fixed tissues respectively. Three of 5 panthers positive by ELISA antigen were also positive by IFA; two (FP109, 115) were inconclusive. Inconclusive results in these panthers may have been due to a delay in testing and/or improper slide storage. Alternatively, if the samples were true negatives,

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30 sampling may have occurred soon after exposure, before infection of the bone marrow. In domestic cats neutropenia or thrombocytopenia can lead to false negatives; however, these values were normal in FP109 and FP115. Spleen and lymph node from FP115 did test positive by IHC when collected 5 mo after initial positive antigen findings. However, tissues from FP109 were severely autolyzed and unsuitable for IHC when collected at necropsy approximately 1 mo after initial positive antigen findings. Epizootiology History of Exposure The FeLV epizootic in free-ranging Florida panthers was foreshadowed by evidence of increasing exposure based on ELISA antibody tests. Beginning in the late 1990s the prevalence of positive ODs increased dramatically, peaking in 2001 when 9 of 26 (34.6%) were positive (Fig. 3-1). Positive ODs were also geographically clustered with 16 of 18 (88.9%) positive ODs occurring north of I-75 (Fig. 3-1). In domestic cats, antibody ODs increase with age; however, this was not seen in panthers probably the result of small sample size. ELISA antibody tests support the theory of multiple introductions of the virus. One introduction may have occurred on the Florida Panther National Wildlife Refuge (FPNWR) between January and November of 2001. Four of five (80%) panthers sampled during the spring of 2001 (2000-2001 capture season) were negative for ELISA antibodies. Two of these panthers (FP96, 99) were recaptured the next season (2001-2002). FP96 had seroconverted from a negative to low positive OD, and FP99 went from a low to medium positive OD. Two other panthers captured in the Fall of 2001 also had positive ODs (FP107, low; FP78, medium). Based on telemetry data, FP96 and 107

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31 (siblings) and FP99 formed a loosely associated group between August and December 2001 (Land et al., 2002). This may have facilitated exposure among these panthers if any were shedding virus at the time. Indeed, one of these, FP96, was found to be latently infected (PCR positive, M. Brown, unpubl. data) at necropsy after being killed by another male in early 2002. Although no panthers from FPNWR tested antigen-positive when sampled, we speculate that FP96 became transiently viremic and exposed the panthers that were accompanying him. He apparently overcame the infection, perhaps aided by a relatively high antibody OD. Prevalence and Distribution Before the 2002-2003 capture season, routine ELISA antigen testing of captured or necropsied Florida panthers had been negative since 1978. However, between July 2002, and June 2005, 5 of 71 (8%) free-ranging panthers/pumas 1 yr of age sampled had active FeLV infections based on ELISA antigen, IFA, and/or viral culture results. All infected panthers had overlapping home ranges in the OKS ecosystem in the north-central portion of panther range (Fig. 3-2). Outcome Following Exposure In domestic cats, prolonged exposure is generally necessary for transmission. Indeed the percentage of adult domestic cats becoming persistently infected following a single exposure event is only 3% (Hartmann, 2005). However, FP132 became infected apparently after an aggressive encounter with an infected panther (FP123). At examination approximately 2 days after the fight, FP132 had only minor scratches and two puncture (presumably bite) wounds. Although FP132 was FeLV antigen negative at this time, he developed a persistent FeLV infection and died 4 mo later. We speculate that bite wounds are an important mode of FeLV transmission in panthers. Feline

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32 leukemia virus is present in highest concentrations (10 6 infectious units/ml) in the saliva (Francis et al., 1977). A relatively larger dose of saliva would be expected to be transferred between fighting panthers versus domestic cats and may explain the apparent ease of transmission. The presence of infection in females is evidence that transmission also may occur during breeding. Males and females will pair for 2 to 5 days and transmission may occur during copulation, mutual grooming, or biting. Although FeLV virus is present in the semen of domestic cats, venereal transmission is not considered important (Hoover and Mullins, 1991). Self-limiting infections In many respects, the outcome following exposure to the virus in panthers is similar to that in domestic cats. Following exposure, a panther can clear the virus early (abortive/transient infections) or can become latently or persistently infected. Based on the relatively large number of panthers with positive ELISA antibody ODs but antigen and PCR negative (M. Brown, unpubl. data) test results, many panthers exposed to the virus are able to clear the infection soon after exposure. Assuming a similar pathogenesis to that occurring in domestic cats, panthers in this category would have cleared the infection within several weeks of exposure before infection of the bone marrow. The majority of domestic cats in this category are considered refractory to re-infection (Hardy, 1980b). Based on telemetry data, at least one female (FP110) with evidence of a previous abortive/transient infection survived exposure to at least two FeLV positive males without developing persistent viremia. It is possible that FP109 was transiently infected when captured in January 2003. At capture he was anemic, lymphopenic, and had a profound lymphadenopathy. Levy

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33 (1999) described similar signs in transiently infected domestic cats. FP109 also had a high ELISA antibody OD. Antibodies detectable by ELISA appear shortly after infection in domestic cats (Lutz et al., 1980b), and high antibody ODs in domestic cats are a good prognostic indicator for recovery (Hofmann-Lehmann et al., 2001). There is also evidence that some panthers can become latently infected as evidenced by positive PCR and antibody ODs but negative ELISA antigen tests. These panthers presumably failed to control infection until later in the course of infection, and retained provirus in leukocytes sufficient to be detectable by PCR. No latently infected panther has developed a persistent infection, and at least one latently infected panther is still surviving in the wild at least 2 yr after diagnosis. Persistent infections In domestic cats, persistent infection is usually characterized by bone marrow infection (positive IFA), viremia persisting for 16 wk, and eventual FeLV-related clinical signs. A diagnosis of persistent infection in panthers was also based on test results, duration of infection, and clinical signs; however, premature deaths, severe autolysis, and limited ability to re-sample panthers while living precluded complete determination of disease progression. Nevertheless, persistent infections were diagnosed in four panthers (FP115, 122, 123, and 132). These diagnoses were based on viremia 16 wk (FP115), positive IFA (FP122, 123, 132), and the presence of FeLV-related diseases (FP115, 122, 123). The latter criterion is speculative; FeLV-infected domestic cats are subject to the same diseases as non-infected cats (Levy, 1999). Nevertheless, the finding of septicemia in two necropsied infected panthers and apparent acute lymphoblastic leukemia in two live-captured infected panthers appears to be unique to those infected

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34 with FeLV. Septicemia without apparent cause or neoplasia was not observed in 73 panther necropsies performed 1978-1999 (Taylor et al., 2002). Persistently infected panthers had relatively low antibody ODs. Although FP115 had a medium positive OD, FP122, 123, and 132 had negative ODs suggesting a muted humoral response to infection. In domestic cats, low ELISA antibody ODs are characteristic of persistent infections (Hoffmann-Lehmann, 2001). Immunosuppression is a common feature of FeLV infection and is believed to result in increased susceptibility to infectious diseases. Co-infections were the most frequent finding in FeLV infected domestic cats examined at North American veterinary schools (Levy, 1999). Infectious and parasitic diseases seen more commonly in FeLV-infected domestic cats than non-infected cats included bacterial infections, hemobartonellosis ( Mycoplasma spp.), FCV, upper respiratory infections, babesiosis, stomatitis, coccidiosis, and toxoplasmosis (Grant et al., 1980; Reinacher, 1989; Reinacher et al., 1995). Of these, bacterial infections, including -hemolytic streptococci, were most important. Jessup et al. (1993) diagnosed septicemia and leptospirosis in a FeLV-infected puma from California. The most significant apparent opportunistic infections in panthers were bacterial; FP115 had an E coli septicemia while a mixture of opportunistic bacteria, predominately -hemolytic streptococci, was cultured from FP132. Other opportunistic infections in viremic panthers may have included M haemofelis and M haemominutum ; however, approximately 70% of FeLV-negative Florida panthers also tested positive for these mycoplasmas (J. Messick, unpubl. data). Feline infectious peritonitis has not been diagnosed in panthers regardless of FeLV status.

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35 Anemias, primarily non-regenerative, are also a frequent finding in FeLV-infected domestic cats. Non-regenerative anemias were seen in FP109 and FP122 when live captured, and may have been the cause of death in FP122. Severe anemia in FP132 was also suspected at necropsy. Bone marrow sections from FP122 and FP132 were examined histologically. Sections were hypercellular with approximately 90% of the marrow space occupied by hematopoietic cells; however, erythroid precursors were decreased in number, and few maturing erythroid cells were present. Finally, hematopoietic neoplasias occur frequently in domestic cats (Reinacher, 1989). Under controlled conditions opportunistic infections can be reduced and neoplasia becomes the most important cause of mortality in FeLV-infected cats (Hofmann-Lehmann et al., 1997). Given the apparently rapid progression of infection, FeLV-positive panthers may not have survived long enough to develop terminal neoplasia. Atypical lymphocytes consistent with acute lymphoblastic leukemia were seen on blood smears from FP122 and FP123; however, no evidence of leukemia was seen on histological examination of bone marrow or other organ tissue from FP122 (FP123 was unsuitable for histological examination). The role, if any, in the deaths of these panthers is unknown. All antigenemic panthers died relatively soon after diagnosis. FP115 and FP132 died from septicemias ( E coli and -hemolytic streptococci respectively). FP122 is believed to have died from severe anemia. FP109 and FP123 died from ISA; however, anemia or other FeLV-related diseases (acute lymphoblastic leukemia) may have impaired their ability to fight. For example, FP109 had a hematocrit of 24% (normal is 36.4% [Dunbar et al., 1997]) when handled 1 mo prior to death, which may have resulted

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36 in exercise intolerance. Although FP123 was apparently healthy when handled 6 wk prior to death, he inflicted only a few minor punctures and scratches to the panther that killed him (FP132). Further, due to severe autolysis, it is unknown if these panthers may have died from secondary bacterial infections. Neither FP109 nor FP123 had obviously fatal ISA-related wounds. Of concern was the presence of healing wounds associated with ISA in FP132 at the time of death suggesting that he may have exposed another panther before dying. Further, these bite wounds may have been the source of infection leading to septicemia in this panther. Progression of infection appears to be quite rapid in panthers. Although 50% of viremic domestic cats die within 6 mo of exposure (Jarrett, 1983), adult cats enjoy a longer induction period and less severe disease compared to younger age groups (Hoover et al., 1976; Levy, 1999). All viremic panthers were adults and, although the time of infection is unknown in most infected panthers, the average time from diagnosis to mortality was just over 9 wk. In the one case of known exposure, FP132 died 18 wk after exposure to an infected male. Lack of supportive care and presumably increased exposure to pathogens may play a role in this apparently more rapid clinical course. Progression to persistent infection following exposure depends on a number of host and viral factors. The most important host factor in domestic cats is age, but inbreeding (genetic variation) and FIV status also affect the outcome. Important viral factors include strain, dose, and duration of exposure. However, with the average age of viremic panthers approaching 5 yr, maturity did not appear to be protective against infection. Genetic variation also did not appear to significantly influence the outcome following exposure. While some panthers had very low genetic variation, at least two had H e much greater

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37 than the average for the population (M. Roelke, unpubl. data). Ancestry also did not appear to play a role. Although three viremic panthers had canonical or original genotypes, two were intergrades (Florida panther/Texas puma and Florida panther/SIR captive) (W. Johnson, unpubl. data). Feline immunodeficiency virus and FeLV, both retroviruses, have overlapping host cell tropism. In domestic cats, co-infection with FIV results in marked synergism of immunosuppression and clinical disease induction (Pedersen et al., 1990). Three of five (60%) FeLV viremic panthers were co-infected with FIV (Shindle et al., 2003; J. Troyer, unpubl. data); however, the impact of co-infection in the panther is unknown. Historically, approximately 28% of the free-ranging panther population was infected with the puma-lentivirus strain of FIV (Olmstead et al., 1992). However, there is a disturbing trend in FIV prevalence 76% (13 of 17) of panthers captured during the 2004-2005 capture season were positive for FIV antibodies. The consequences of this trend are unknown but if pre-existing FIV infection affects subsequent FeLV infection, then an increasing FIV prevalence may alter the epizootiology of FeLV infection in panthers. Viral factors may play a more important role in the apparent greater impact of FeLV on panthers. Although infection pressure is expected to be low in this reclusive solitary species, viral load may be quite high. Although the virus concentration in saliva of infected panthers is unknown, the dose transmitted during fighting or breeding would be expected to be higher as panthers may be as much as 15-fold larger than domestic cats. However, virus strain may be the most important factor in the current epizootic. Based on

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38 preliminary viral culture results, the strain isolated from the current epizootic in panthers may be similar to virulent domestic cat strains (K. Hayes, unpubl. data). Epizootiology The source of infection in panthers is unknown. Texas pumas introduced in 1995 tested antigen negative during quarantine (Dunbar, 1995), and western pumas in captivity at SIR tested negative when handled in 1999 (Shindle et al., 2000). In reports of FeLV infection in non-domestic felids, authors speculated or provided direct evidence that an infected domestic cat was the source and this is the most likely explanation for FeLV in panthers. Domestic cat remains have been found in the stomachs of necropsied pumas from California (Jessup et al., 1993) and there have been observations of panthers killing domestic cats in Florida (L. Richardson, pers. commun.). Kennedy-Stoskopf (1999) speculated that [c]onsumption of FeLV infected domestic cats by larger nondomestic felids wouldbe an effective way to transmit the virus. Private land in panther range continues to be developed at an astounding pace. As humans encroach on panther habitat they are accompanied by their domestic animals, including cats. Indeed free-roaming domestic cats have been observed on private lands near OKS (M. Lotz, pers. commun.). Additionally, the increasing panther population undoubtedly results in increased opportunities for exposure to domestic cats. Young dispersing males move through the fringes of the resident population and often occupy marginal habitat until an established home range becomes available (Maehr, 1997). This existence on the urban/wildland interface likely increases the risk for exposure to domestic cats. Riley et al. (2004) speculated that a higher prevalence of positive feline calicivirus titers in bobcats frequenting urban areas was due to increased exposure to domestic cats.

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39 The transmission of FeLV from a domestic cat to a panther is likely a rare event. Domestic cat remains have never been reported in panther scat (Maehr et al., 1990) or in stomach contents (M. Cunningham, unpubl. data). Further, the odds of a free-ranging domestic cat in Florida having FeLV is less than 1 in 20 (Lee et al., 2002). Should these unlikely events occur the panther would still need to become persistently infected following exposure. Given that these events, however unlikely, probably did occur, once the species barrier was crossed the virus was likely spread panther-to-panther. The apparent transmission of FeLV from FP123 to FP132 supports this theory. Higher panther densities undoubtedly facilitate this panther-to-panther transmission. The population has tripled since the early 1990s while panther habitat has been reduced. Feline leukemia virus infection in panthers is likely a disease of adult cats. Although mother-to-offspring transmission is probably the most important mode of transmission in domestic cats (Levy, 2005) it is unlikely to be a factor in the epizootiology of the disease in panthers. Given the apparent severity and rapid progression of the disease in panthers, infected females are unlikely to survive to raise kittens. Further, if a female were to successfully reproduce, infected kittens would be unlikely to survive to independence the age at which an infected kitten would first be expected to encounter susceptible panthers and potentially spread the virus. The current epizootic likely began in the OKS area in late 2001 or early 2002, possibly as a result of cross-species transmission from an infected domestic cat on a local ranch. The first positive antibody tests in OKS began to appear in February 2002. The infection was then likely spread panther-to-panther resulting in the infection of at least

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40 five panthers. However, since July 2004, none of 30 panthers have tested FeLV antigen-positive indicating that the epizootic may be over. Several factors may have contributed to this. First, the rapid progression of disease may have limited the number of exposure events infected panthers die before transmitting the disease. Additionally, some panthers appear to be refractory to infection thus limiting the number of susceptible individuals capable of perpetuating the disease. Small population size and geography may have also helped. Fromont et al. (1998a) demonstrated that populations less than 100 individuals were unlikely to sustain FeLV infections. Since this epizootic occurred in the northernmost portion of panther range the disease could effectively spread only to the south. Finally, vaccination may have helped end the epizootic. Vaccination of free-ranging panthers began in August 2003, and as of June 2005, 34 panthers have received at least one inoculation. Six of these have died due to non-FeLV causes, therefore, based on a population size of between 80 and 100, approximately 28 to 35% of the population has received at least one inoculation. However, because vaccination efforts were targeted at OKS and adjacent lands, the percentage vaccinated in these areas is much greater. Using computer models, Lubkin et al. (1996) estimated that 23% to 73% of a population with a FeLV prevalence of 10% must be effectively vaccinated to eliminate infection. Conclusion Kennedy-Stoskopf (1999) speculated [t]he lack of antigen-positive animals and absence of clustered clinical cases with FeLV-related diseases are evidence that the virus is not maintained in [non-domestic felid] populations. However, the finding of five antigenemic panthers over almost 2 yr is evidence that the disease had, at least temporarily, become established in the Florida panther population. Small populations are at greater risk of extinction due to infectious diseases than larger populations

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41 (Berger,1990). With the exception of the occasional dispersing male, all free-ranging Florida panthers are part of a single contiguous population in south Florida. As such, the population is at risk of a catastrophic disease outbreak. Also of concern is the apparent increased FeLV susceptibility of panthers compared to domestic cats. Transmission appears to be occurring despite low infection pressure (few exposure events) and host maturity, and the progression of infection appears to be more rapid compared to domestic cats. Finally, FeLV prevalence among free-ranging domestic cats in Florida is <4% (Lee et al., 2002), and in a review of FeLV in domestic cats, Levy (1999) speculated that [t]rue outbreaks of FeLV infection are unlikely to occur. However, the prevalence of FeLV in panthers sampled in OKS went from 0% prior to 2002 to 45.5% between November 2002, and June 2005. If the disease spreads to the core population the impact could be devastating. Further Research More research is needed to further elucidate the epizootiology of FeLV in panthers. Western blot antibody tests are needed to confirm positive ELISA antibody tests. Further, quantitative PCR may be used to estimate provirus burden in panthers. This technique may be more sensitive than conventional PCR for detecting latent infections. (Hofmann-Lehmann et al., 2001). In domestic cats latent infections may eventually be cleared. Testing to determine if living PCR-positive panthers converted to negative status should be performed to determine the duration of the latent state. Finally, further research is needed to determine the source of infection. Bobcats should be tested in the OKS region. Domestic cats from this area should be tested as well, and virus recovered from infected cats should be sequenced for comparison to strains isolated from panthers.

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APPENDIX A FLORIDA PANTHER/TEXAS PUMAS SAMPLED DURING THE STUDY PERIOD

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Table A-1. Florida panthers and Texas pumas tested for feline leukemia virus (FeLV) antigen by ELISA 1 July 2002, to 5 June 2005. FeLV FIV ID a Date Event b Sex c Age (yr) Location d Area e Antibody OD f Antibody result g ELISA antigen h Western Blot i FP 32 9/12/2002 N F 15 FPNWR North N FP 48 2/18/2005 L F 10.25 BCNP-N North 0.227 N N P FP 55 4/4/2003 L F 10 BCNP-C South 0.188 N N P FP 59 4/20/2004 L M 9.33 FSSP South 0.176 N N E FP 59 11/22/2004 N M 9.42 PL-Collier Co. North N FP 60 6/29/2004 L M 8.75 BCNP-S South 0.219 N N P FP 67 1/15/2003 N F 5.5 PL-Collier Co. North N FP 69 1/3/2005 L F 7.75 BCNP-N North 0.252 L N P FP 70 3/10/2003 L F 5.83 BCNP-C South 0.131 N N N FP 71 2/18/2004 L F 6.75 SIR North 0.173 N N N FP 73 2/27/2003 L F 6 BCNP-N North 0.17 N N N FP 75 5/6/2003 L F 5 BCNP-N North 0.184 N N N FP 78 10/16/2002 N F 6 FPNWR North N FP 79 3/17/2004 L M 8.5 BCNP-C South 0.116 N N N FP 79 3/3/2005 L M 9.5 BCNP-C South 0.176 N N P FP 82 12/6/2002 L F 6 OKS North 0.262 L N N FP 83 3/31/2004 L F 4.8 FSSP South N FP 85 2/18/2003 L M 4 ENP South 0.165 N N N FP 85 3/1/2004 N M 5 ENP South N FP 86 4/1/2003 L F 3.92 BCNP-C South 0.165 N N N FP 86 11/6/2003 L F 3.92 BCNP-C South 0.094 N N N FP 91 3/18/2003 L F 3.75 BCNP-C South 0.216 N N N FP 91 12/12/2003 N F 4.5 BCNP-C South N FP 94 1/30/2003 L F 3.5 ENP South 0.141 N N N FP 95 3/24/2003 L F 5.17 ENP South 0.168 N N N FP 98 7/1/2002 N M 4 SR29 North N FP 99 11/27/2002 N M 2.8 CR846 North N FP 100 1/6/2004 L M 7 BCNP-N North 0.155 N N N 43

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44 Table A-1. Continued. FeLV FIV ID a Date Event b Sex c Age (yr) Location d Area e Antibody OD Antibody result f ELISA antigen g Western Blot h FP 102 3/24/2004 L F 6 BCNP-C South 0.121 N N N FP 103 2/27/2004 L F 3.75 BCNP-C South 0.143 N N N FP 104 12/13/2002 L M 2.13 BCNP-N South 0.132 N N P FP 104 3/1/2005 L M 5.4 BCNP-C South 0.133 N N P FP 106 2/20/2003 N F 3 SR29 North N FP 107 12/6/2004 L F 4.7 FPNWR North 0.23 N N P FP 108 11/17/2002 N M 1.9 BCNP-C South N FP 109 1/24/2003 L M 11 OKS North 0.546 H P N FP 109 2/21/2003 N M 11.08 OKS North N j FP 110 11/25/2002 L F 1.08 OKS North 0.196 N N E FP 112 9/11/2002 N F 4 BCNP-N North N FP 113 10/23/2002 L F 0.54 FPNWR North 0.183 N N P FP 113 k 4/7/2004 L F 2 FPNWR North 0.237 N N E FP 114 10/23/2002 L M 0.54 FPNWR North 0.242 N N P FP 114 k 10/17/2003 N M 2.5 FPNWR North N FP 115 11/26/2002 L F 4.5 OKS North 0.499 M P P FP 115 5/17/2003 N F OKS North P FP 116 1/20/2003 L F 0.63 PL-Hendry Co. North 0.149 N N N FP 116 k 3/22/2004 L F 1.8 PL-Collier Co. North 0.142 N N N FP 117 2/25/2003 L M 0.88 BCNP-N North 0.17 N N N FP 117 12/3/2003 L M 1.58 SIR North 0.157 N N N FP 117 k 7/28/2004 N M 2.25 OKS North N FP 118 3/5/2003 L F 0.92 SIR North 0.157 N N N FP 118 4/2/2003 N F 1 SIR North N FP 119 4/2/2003 L M 1 BCNP-C South 0.125 N N P FP 119 11/17/2004 L M 2.7 FPNWR North 0.237 N N P

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45 Table A-1. Continued. FeLV FIV ID a Date Event b Sex c Age (yr) Location d Area e Antibody OD Antibody result f ELISA antigen g Western Blot h FP 120 4/8/2003 L F 3 BCNP-C South 0.193 N N N FP 120 7/14/2004 L F 4 BCNP-C South 0.163 N N P FP 121 12/2/2003 L F 2.5 SIR North 0.148 N N N FP 122 1/30/2004 L F 2.25 OKS North 0.183 N P N FP 122 2/13/2004 N F 2.25 OKS North P FP 123 2/2/2004 L M 3.5 OKS North 0.151 N P N FP 123 3/15/2004 N M 3.6 OKS North P FP 124 2/13/2004 L F 3.5 BCNP-S South 0.155 N N P FP 125 2/13/2004 L M 0.67 BCNP-S South 0.135 N N N FP 126 2/13/2004 L M 0.67 BCNP-S South 0.155 N N N FP 126 k 5/28/2004 L M 0.96 BCNP-S South 0.163 N N FP 127 2/16/2004 L M 2 BCNP-C South 0.149 N N P FP 127 k 3/29/2005 L M 3 BCNP-N South 0.196 N N P FP 128 2/18/2004 L F 3.7 SIR North 0.171 N N N FP 129 2/20/2004 L F 3 BCNP-C South 0.157 N N P FP 130 3/4/2004 L M 0.8 OKS North 0.181 N N N FP 130 k 3/10/2005 L M 1.83 PL-Highlands Co. North 0.385 M N P FP 131 3/10/2004 L M 5 FPNWR North 0.193 N N N FP 132 3/17/2004 L M 3 OKS North 0.108 N N N FP 132 7/22/2004 N M 3.33 OKS North 0.23 N P N FP 133 11/18/2004 L M 4.5 BCNP-N North 0.214 N N P FP 134 12/14/2004 L M 2.5 BCNP-N North 0.246 N N P FP 135 12/17/2004 L M 1.7 FPNWR North 0.24 N N N FP 136 1/13/2005 L F 5 BCNP-C South 0.207 N N N FP 137 1/25/2005 L M 2.5 OKS North 0.239 N N N FP 138 1/31/2005 L M 4 BCNP-C South 0.212 N N P FP 139 3/31/2005 L M 2.83 OKS North 0.213 N N N

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46 Table A-1. Continued. FeLV FIV ID a Date Event b Sex c Age (yr) Location d Area e Antibody OD Antibody result f ELISA antigen g Western Blot h K 94 8/17/2004 N M 3 I75 North-South N K 149 6/11/2003 L F 0.05 OKS North N K 150 6/11/2003 L M 0.05 OKS North N K 156 8/2/2004 N M 0.5 US41 South N K 175 2/10/2005 L M 0.04 BCNP-S South N K 176 2/10/2005 L M 0.04 BCNP-S South N K 178 3/7/2005 L M 0.05 OKS North N K 179 3/7/2005 L F 0.05 OKS North N K 180 3/21/2005 L F 0.05 FPNWR North N K 181 3/21/2005 L F 0.05 FPNWR North N TX 105 1/27/2003 L F 10.5 ENP South 0.161 N N P TX 106 1/8/2003 L F 9.5 PSSF South 0.129 N N P TX 108 11/18/2002 L F 10 ENP South 0.189 N N N

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47 Table A-1. Continued. FeLV FIV ID a Date Event b Sexc Age (yr) Location d Area e Antibody OD Antibody result f ELISA antigen g Western Blot h UCFP 48 11/11/2002 N F 0.7 CR846 North N UCFP 49 11/25/2002 N F 1.58 PL North N UCFP 50 1/25/2003 N M 6.5 PL North N UCFP 51 3/10/2003 N M 2 NC North N UCFP 54 6/3/2003 N M 0.75 PL North N UCFP 58 6/30/2003 N F 0.83 PL North N UCFP 59 11/2/2003 N M 0.3 CR858 North N UCFP 60 12/9/2003 L M 1.5 BCNP South 0.144 N N N UCFP 61 12/25/2003 N F 2.5 CR833 North N UCFP 62 1/11/2004 N F 0.63 BCNP-C-S South N UCFP 63 2/26/2004 N M 3.5 I75 North-South 0.166 N N UCFP 65 4/6/2004 N M 1.5 SR29 North N UCFP 66 6/27/2004 N M 1.75 I75 North N UCFP 67 9/2/2004 N F 0.003 FPNWR North N UCFP 68 9/28/2004 N F U BCNP-C South N UCFP 69 10/25/2004 N F 2 SR29 North N UCFP 70 12/1/2004 N F 1 SR29 North N UCFP 71 2/4/2005 N M 2.5 US41 South N UCFP 74 6/4/2005 N M 3 I95 Flagler Co. North N

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48 TABLE A-1 LEGEND a FP (Florida panther), TX (Texas puma), K (Florida panther previously handled as a neonatal kitten), UCFP (Uncollared Florida panther). b L (live-capture), N (necropsy) c F (female), M (male). d BCNP (Big Cypress National Preserve C [BCNP between I-75 and US-41], N [BCNP north of I-75], S [BCNP south of US-41]), CR (County Road), CWMA (Crew Wildlife Management Area), ENP (Everglades National Park), FPNWR (Florida Panther National Wildlife Refuge), I (Interstate), OKS (Okaloacoochee Slough State Forest), PL (private lands), PSSF (Picayune Strand State Forest), SIR (Big Cypress Seminole Indian Reservation), SR (State Road), US (United States Road). e N (panther range north of I-75), S (panther range south of I-75), N-S (killed by vehicular collision on I-75). f OD (optical density). g N (negative), L (low positive), M (medium positive), H (high positive). h N (negative), P (positive). i N (negative), P (positive), E (equivocal). j Test results unsuitable due to severe autolysis. k Previously vaccinated for feline leukemia virus.

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APPENDIX B CASE REPORTS: ANTIGENEMIC FLORIDA PANTHERS FP115 On 26 November 2002, a 4.5 yr-old female Florida panther was captured in OKS. Capture was routine and the panther appeared healthy weighing 52.7 kg. Physical exam was unremarkable and routine biomedical samples were collected. The panther was vaccinated with Fel-O-Vax PCT, de-wormed, radio-instrumented and released. Complete blood count revealed a mild non-regenerative anemia (28.4%), low hemoglobin (9.2 g/dL), and lymphopenia (736/l). Biochemical alterations were limited to an elevated BUN (57 mg/dL), glucose (183 mg/dL), and creatinine phosphokinase (609 U/L). FeLV ELISA antigen and FIV ELISA antibody (KELA and Western Blot) were positive, although IFA of blood smears were inconclusive. Feline leukemia virus was cultured from EDTA whole blood at the Ohio State University (OSU). Radio-telemetry over the ensuing 5 mo indicated normal movements with a minimum convex polygon (MCP) home range of 104.6 km 2 (Shindle et al., 2003). However, between 12 and 17 May 2003 movements became increasingly restricted. On 17 May 2003, the panther was located in a palmetto thicket and died at approximately 1730 hrs. The panther was immediately collected, placed on ice, and transported to Disneys Animal Kingdom the next day for complete necropsy. The panther had lost 20.4 kg since capture; moderate SQ and abdominal fat were present although there was mild muscle wasting. A copious red-tinged mucosy fluid drained from the nares. The right 49

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50 submandibular lymph node exuded a bloody purulent yellow fluid. The tracheobronchial lymph nodes were markedly enlarged and had a nodular appearance on cut section. A frothy yellow mucoid material was observed at the tracheal bifurcation extending into the distal airways. The lungs had a diffusely mottled red/black appearance with a nodular texture which extended into the cut surface. The caudal lung lobes were most severely affected. The thymus contained multifocal hemorrhages, and the pericardial sac contained fibrin. The mesenteric lymph nodes were diffusely prominent. The stomach contained a small amount of hog hair and mucus. The spleen was slightly enlarged and had a meaty texture. The liver contained 1-3 mm multifocal tan foci. Each ovary contained two 3-5 mm diameter corpora lutea, and the uterus showed no evidence of previous pregnancy. Histologically, the lungs contained nodular collections of alveoli containing dense colonies of gram-negative bacteria intermixed with degenerative neutrophils, fibrin, extravasated erythrocytes, and necrotic debris. There was necrosis of type I pneumocytes and scattered hyperplasia of type II pneumocytes. Adjacent alveoli contained edema fluid, macrophages, and neutrophils. Within the kidneys there was multifocal mineralization of cortical tubules associated with necrosis of tubular epithelial cells. The tubular epithelial cells contained a golden brown granular pigment. Throughout the splenic parenchyma there was a mild increase of macrophages with mild hyperplasia of white pulp. Megakaryocytes were scattered throughout the red pulp. Fibrin was seen in a few splenic sinuses. Within the submandibular lymph node there was a focally extensive area of necrosis infiltrated by large numbers of degenerate neutrophils. Other lymph nodes showed evidence of multifocal cortical hyperplasia with sinuses containing

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51 macrophages and lymphocytes. All other tissues appeared histologically normal. Immunohistochemistry of spleen and lymph node was positive for p27 antigen. Aerobic culture of the lung, liver, and submandibular lymph node resulted in pure growth of E coli Viral isolation of the lung was negative. Although IFA was inconclusive at capture, the persistence of antigenemia for >5 mo combined with clinical signs is consistent with persistent infection. FP109 FP109 was initially captured and radio-instrumented as a 10 yr-old male 10 February 2002, in OKS. At capture he had injuries consistent with intraspecific aggression. ELISA ag for FeLV at Cornell University was negative as was ELISA antibody (HVL). Ten days later FP109 had to be recaptured to replace a defective radio-collar and ELISA ag was again negative. The radio-collar failed several wk later. FP109 was recaptured 24 January 2003, at 11 yr of age. At capture he appeared to be in excellent condition but had a pronounced peripheral lymphadenopathy. Benign hyperplasia was diagnosed from fine-needle aspirates of the popliteal lymph nodes. Complete blood count revealed a moderate non-regenerative anemia (23.8%), low hemoglobin (7.5 g/dL), and lymphopenia (490/l). Rare C felis organisms were seen on blood smears. Serum biochemical abnormalities were minor with only an elevated glucose (190 mg/dL) and decreased triglycerides (10 mg/dL). Feline leukemia virus ELISA antigen and antibody tests were positive although IFA of blood smears were inconclusive. Feline leukemia virus was cultured from EDTA whole blood at OSU. Radio-telemetry indicated normal movements, but on 27 February 2003, FP109 was found dead. The panther had been dead for 2-3 days, and the carcass was severely autolyzed, decomposed, and partially scavenged. Partial necropsy revealed puncture

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52 wounds in the skin over the nasal bones with symmetrical crushing fractures of the nasal bones. Injuries were consistent with ISA. FP109 died before his true FeLV status could be determined. At capture in January 2003 he was likely in the early stages of infection. Given his high antibody OD, it is possible he may have eventually cleared the infection. Negative ELISA antigen findings in heart blood collected at necropsy supports this speculation; however, the sample was extremely autolyzed and should be considered unreliable. FP122 On 30 January 2004, a 2.25 yr-old female Florida panther was captured in OKS. Capture was routine and the panther appeared in poor health weighing only 32.3 kg with minimal SQ fat. Physical exam was otherwise normal except for a peripheral lymphadenopathy. Routine biomedical samples were collected, and a SNAP test using EDTA whole blood in the field was positive. The panther was vaccinated, de-wormed, radio-instrumented, and released. Complete blood count revealed a moderate non-regenerative anemia (22.5%), low hemoglobin (7.2 g/dL), and monocytosis (1020/l). Mild polychromasia, mild to moderate anisocytosis, and 10 nucleated red blood cells/100 leukocytes were seen on peripheral blood smear. Additionally, large immature mononuclear cells that occasionally contained nucleoli were also seen; these findings were interpreted as an acute lymphoblastic leukemia. Serum biochemical abnormalities included low cholesterol (62 mg/dL) and triglycerides (9 mg/dL). Abnormalities seen on urinalysis of free-catch urine included 1+ blood, 3-10 WBC/HPF, 1-3 RBC/HPF, and 4+ bacteria/HPF. Specific gravity was 1.009. ELISA antigen and IFA were positive. Virus was cultured from EDTA blood at OSU.

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53 Radio-telemetry over the ensuing 2 wk indicated normal movements and she remained within the OKS area. Approximately 1 wk after capture, field sign indicated the panther had killed and fed on a white-tailed deer ( Odocoilius virginianus ). However, on 13 February 2004, a mortality signal was detected, and her carcass was found in a hammock in OKS. Time of death was approximately 0500 hrs. The panther was immediately collected, placed on ice, and transported to Disneys Animal Kingdom the next day for complete necropsy. At necropsy the panther was approximately 15% dehydrated, in poor body condition, and had lost 8.9 kg since capture. Mucus membranes and skeletal muscle were pale. Abdominal and SQ fat were negligible, and there was evidence of serous fat atrophy. Adrenal glands were diffusely enlarged. Peripheral lymph nodes were markedly enlarged. Histologically the bone marrow was hypercellular with approximately 90% of the marrow space occupied by hematopoietic cells. There was also a moderate increase in the number of megakaryocytes. Erythroid precursor cells were decreased in number, and few maturing erythroid cells were present. Myeloid cell lines were relatively increased in number, and all stages of maturation were observed. Few lymphoid precursors and mature lymphocytes were seen, and there was no marrow evidence of an acute leukemia. There was no evidence of cortical follicle formation in examined lymph nodes. Thymocytes were present in the thymus, but there was no evidence of cortical or medullary architecture; intermixed among the thymocytes were macrophages containing a bland golden brown pigment. Alveoli contained eosinophilic fluid and mildly increased numbers of alveolar macrophages. Within the spleen, much of the red pulp was autolyzed and there was scattered extramedullary hematopoiesis evident with few megakaryocytes

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54 noted. Within the kidneys, scattered glomeruli were shrunken with markedly thickened Bowmans capsules, collapse of the glomerular tufts, and replacement of the glomerular tufts by fibrillar eosinophilic material. Rare cortical tubules were dilated. Scattered few perivascular infiltrates of lymphocytes and plasma cells were present in the cortical interstitium. Rabies IFA was negative. Brain and heart were negative for CDV, pseudorabies virus, flaviviruses, and alphaviruses by real-time and conventional PCR and viral culture. Persistent infection was diagnosed based on positive ELISA antigen and IFA results and clinical signs. FP123 FP123, a 3.5 yr-old male, was captured 2 February 2004, in OKS. Capture was routine and the panther appeared healthy weighing approximately 64 kg. Physical exam was unremarkable and routine biomedical samples were collected. FeLV SNAP test using EDTA whole blood was positive. The panther was vaccinated, dewormed, radio-instrumented and released. Complete blood count abnormalities were limited to a lymphopenia (884/l), although a significant percentage (11%) of the differential contained large, apparently immature, mononuclear cells that occasionally contained nucleoli. These findings were interpreted as an acute lymphoblastic leukemia. Serum biochemical abnormalities were suggestive of dehydration and recent feeding (BUN 59 mg/dL, sodium 161 mEq/L, BUN/creatinine ratio 39, triglycerides 222 mg/dL, and calculated osmolality 336 mOsm/L). FeLV ELISA antigen at Cornell Diagnostic Laboratory and IFA of blood smears at the National Veterinary Laboratory were positive.

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55 FP123 had a large home range (164.5 km 2 ) traveling at least 10 km to the south (Shindle et al., 2004). However, within 6 wk of capture, FP123 was found dead 17 March 2004, in OKS following detection of a mortality signal. The carcass was severely autolyzed and decomposed; date of death was believed to have been 15-16 March. FP132 was captured the same day within 400 m of FP123. Acute injuries on FP132 were consistent with ISA. FP123 was completely necropsied at Disneys Animal Kingdom. No gross abnormalities were noted although the carcass was severely autolysed. FP132 On 17 March 2004, the carcass of FP123, a FeLV positive male, was recovered in OKS. External injuries indicated the cause of death to be intraspecific aggression, and the panther appeared to have been dead for approximately 24-48 hrs. Within 400 m of the carcass, a freshly killed white-tailed deer was discovered. The dogs were released and FP132, a 3 yr-old male, was captured. Capture was routine and the panther appeared healthy weighing 66.3 kg. Two acute puncture wounds over the right shoulder, presumably bite wounds, were seen on physical examination. Minor lacerations consistent with claw marks were also seen. Circumstantial evidence was consistent with FP132 as the cause of death for FP123. Routine biomedical samples were collected and a SNAP test using EDTA whole blood in the field was negative. The panther was vaccinated (including 2 ml Fel-O-Vax LvK), de-wormed, radio-instrumented, and released. Complete blood count and serum chemistry were unremarkable. Repeat FeLV ELISA antigen test was negative.

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56 Radio-telemetry over the ensuing 4 mo indicated normal movements with a home range of approximately 197.4 km 2 (Shindle et al., 2004). FP132 was treed and boostered with 2 ml Fel-O-Vax LvK on 12 April 2004. However, detectable movement based on radio telemetry ceased between 14 and 21 July 2004. On 20 and 21 July, biologists investigated and were able to approach to within 5 m of FP132 in thick brush before he would move ahead. He appeared alert and healthy but lethargic. On 22 July, the panther was located in a palmetto thicket and appeared to be in respiratory distress. He died at approximately 1000 hr. Within 30 min of death, whole blood was collected by dissection of the brachial artery and aspiration with a needle and syringe. Blood was placed in serum separator and EDTA tubes, and blood smears were made from cells (EDTA). The panther was transported from the field and was on ice within 3 hr of death. Necropsy was performed at Disneys Animal Kingdom. At necropsy the panther was approximately 10% dehydrated and had lost over 13 kg since capture. There was moderate muscle wasting although moderate to heavy SQ and abdominal fat was present. A 15x15 mm pedunculated cutaneous mass was present over the left nasomaxillary region. Mucus membranes were icteric and pale, and a copious red-tinged fluid drained from the nares. Several healing puncture wounds and abrasions were noted in multiple sites. Gross examination revealed a large abscess occupying the subcutis over the lateral aspect of the right quadriceps muscle. The abscess measured 29x17 cm and had a variable depth of 5-10 cm. The abscess contained several liters of tan cloudy fluid. Skeletal muscle was pale. Lungs were diffusely dark red and firmer than expected. On cut section numerous 1-5 mm tan foci were observed in all lung lobes although the left

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57 cranial and medial lobes were most severely affected. Sections of lung tissue from these lobes did not float in formalin. The liver was pale and friable. Peripheral lymph nodes were not significantly enlarged, but mesenteric lymph nodes were larger than expected. Aerobic cultures were taken of the abscess and lungs resulting in heavy growth of -hemolytic Streptococcus sp. Histologically, the skeletal muscle beneath the abscess was covered by a thick band of mixed inflammatory cells representing the margin of the abscess. The superficial aspect was composed of large numbers of degenerate neutrophils subtended by mixed macrophages, lymphocytes, and plasma cells as well as immature fibroblasts and connective tissue markedly expanded by edema. Numerous colonies of large bacterial cocci were present on the superficial aspect of the lesion. Multifocally throughout the lung, large dense colonies of bacteria and associated inflammatory cell aggregates effaced the pulmonary architecture. Smaller bacterial colonies were also common in airways. Large numbers of degenerate neutrophils and alveolar macrophages were present in association with bacterial colonies and within the adjacent parenchyma. There was necrosis of alveolar epithelium with multifocal type II pneumocyte hyperplasia. Alveoli often contained strands of fibrin and edema fluid. Large areas of necrosis and hemorrhage were also present. Numerous small (approx 50um diameter) objects resembling trematode eggs were scattered throughout the liver. These eggs were bounded by a refractile rim with the central core composed of granular basophilic material. Numerous eggs were mineralized, degenerate, and were associated with small numbers of macrophages. Within the thymus there was a loss of architecture and replacement by abundant adipose tissue. There was also loss of cortico-medullary demarcation with

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58 lymphocytes remaining in a loosely arranged fibrovascular stroma. Scattered cystic structures were present and were presumed to be Hassals corpuscles. Also present within the thymus were small mineralized structures which also represented calcified Hassals corpuscles. Increased numbers of large macrophages containing pale brown cytoplasmic pigment were present in the thymic parenchyma. Sections of bone marrow were hypercellular with approximately 100% of the marrow space occupied by hematopoietic cells. Megakaryocytes were present in normal to mildly increased numbers. Serum biochemical abnormalities were consistent with hepatic failure (total bilirubin 5.8 U/L, ALT 455 U/L, and AST 728 U/L) and pre-renal azotemia and/or renal failure (BUN 63 mg/dL, creatinine 2.8 mg/dL). Other evidence of renal failure included a severe hyperkalemia (9.7 mEq/L), hyperphosphotemia (19.1 mEq/L), hypermagnesemia, and calculated osmolality (338 mOsm/L). These findings were likely a combination of post-mortem artifact (potassium released from platelets), tissue necrosis, metabolic acidosis, and dehydration. Hypoglycemia (61 mg/dL) was likely the result of septicemia. FeLV SNAP test of serum and aqueous humor, and IFA of blood smears were positive. Immunohistochemistry of spleen and lymph node were positive for p27 antigen. ELISA antigen of serum at Antech Diagnostics was negative, but this is believed to be an erroneous result. Virus was cultured at OSU.

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59 Table B-1. Selected hematological and serum biochemical values for Florida panthers testing positive for feline leukemia virus (FeLV) antigen by ELISA 1 July 2002 to 5 June 2005. Panther FP 109 FP 115 FP 122 FP 123 FP 132 Units Normal b (SD) c Sex M F F M M Date 1/24/2003 11/26/2002 1/30/2004 2/2/2004 7/22/2004 Age (yr) 11 4.5 2.25 4 3 FIV e western blot N P N N N FeLV ELISA P P P P P FeLV IFA f blood smear N N P P Glucose 190 183 115 120 61 mg/dl 154.4 (51.0) Blood urea nitrogen 22 57 24 59 63 mg/dl 37.7 (14.1) Creatinine 2.0 2.1 2.4 1.5 2.8 mg/dl 1.84 (0.54) Total protein 8.0 6.6 6.5 7.4 5.5 g/dl 7.35 (0.67) Albumin 3.4 3.2 3.3 3.6 2.7 g/dl 3.70 (0.36) Bilirubin 0.1 0.2 0.3 0.3 5.8 mg/dl 0.26 (0.61) Alkaline phosphatase 7 12 8 7 4 U/l 35.4 (38.6) Alanine aminotransferase 45 36 35 62 455 U/l 60.2 (35.0) Aspartate aminotransferase 24 36 28 68 728 U/l 73.4 (77.8) Calcium 10.4 9.3 10.3 9.8 10.2 mg/dl 9.92 (0.66) Phosphorous 3.6 4.8 5.0 5.2 19.1 mg/dl Globulin 4.6 3.4 3.2 3.8 2.8 g/dl Creatine phosphokinase 223 609 244 470 223 U/l 515.6 (415.1) Hemoglobin 7.5 9.2 7.2 13.2 g/dl 12.21 (1.70) Hematocrit 23.8 28.4 22.5 42.5 % White blood cells 4.9 9.2 8.5 5.2 X103/l 12.19 (3.01) Red blood cells (RBC) 4.91 6.85 4.18 8.75 X106/l 7.635 (1.033) Mean cell volume 48 41 54 49 47.29 (2.89) Mean cell hemoglobin (MCH) 15.3 13.4 17.2 15.1 Pg 16.07 (1.41) MCH concentration 31.5 32.4 32.0 31.1 g/dl 34.08 (3.26) Platelets 147 236 185 350 X103/l 402.6 (131.5) Neutrophils 4165 7360 4845 3848 8000 (2900) Neutrophils % 85 80 57 74 % WBC's 64.3 (14.3) Band 0 0 0 0 Band % 0 0 0 0 % WBC's Lymphocytes 490 736 2550 884 3400 (1700) Lymphocytes % 10 8 30 17 % WBC's 28.8 (14.5) Monocytes 245 368 1020 416 390 (340) Monocytes % 5 4 12 8 % WBC's 3.2 (2.6) Eosinophils 0 736 85 52 420 (310) Eosinophils % 0 8 1 1 % WBC's 3.4 (2.2) Basophils 0 0 0 0 100 (60) Basophils % 0 0 0 0 % WBC's 0.89 (0.57) Nucleated RBC's (NRBC) 0 10 0 /100WBC's 1.5 (1.0) T4 1.3 1.6 1.3 0.2 0.2 g/dl Cytauxozoon P N N N a ELISA (Enzyme-linked immunosorbent assay). b Normal values for panthers (Dunbar et al., 1997). c SD (standard deviation). d OKS (Okaloacoochee Slough). e FIV (feline immunodeficiency virus). f IFA (immunofluorescent assay).

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LIST OF REFERENCES BARONE, M. A., M. E. ROELKE, J. HOWARD, J. L. BROWN, A. E. ANDERSON, AND D. E. WILDT. 1994. Reproductive characteristics of male Florida panthers: Comparative studies from Florida, Texas, Colorado, Latin America, and North American zoos. Journal of Mammalogy 75: 150-162. BEEBE, A. M., T. G. FAITH, E. E. SPARGER, M. TORTEN, N. C. PEDERSEN, AND S. DANDEKAR. 1994. Evaluation of in vivo and in vitro interactions of feline immunodeficiency virus and feline leukemia virus. AIDS 8: 873-878. BERGER, J. 1990. Persistence of different-sized populations: an empirical assessment of rapid extinctions in bighorn sheep. Conservation Biology 4: 91-98. BIEK, R., R. L. ZARNKE, C. GILLIN, M. WILD, J. R. SQUIRES, AND M. POSS. 2002. Serologic survey for viral and bacterial infections in western populations of Canada lynx ( Lynx canadensis ). Journal of Wildlife Diseases 38: 840-845. BRIGGS, M. B., AND R. L. OTT. 1986. Feline leukemia virus infection in a captive cheetah and the clinical and antibody response of six captive cheetahs to vaccination with a subunit feline leukemia virus vaccine. Journal of the American Veterinary Medical Association 189: 1197-1199. CITINO, S. B. 1986. Transient FeLV viremia in a clouded leopard. Journal of Zoo and Wildlife Medicine 17: 5-7. COURCHAMP, F., C. SUPPO, E. FROMONT, AND C. BOULOUX. 1997. Dynamics of two feline retroviruses (FIV and FeLV) within one population of cats. Proceedings of the Royal Society of London B 264: 785-794. CUNNINGHAM, M. W., M. R. DUNBAR, C. D. BUERGELT, B. L. HOMER, S. K. TAYLOR, R. KING, S. B. CITINO, C. GLASS, AND M. E. ROELKE-PARKER. 1999. Atrial septal defects in Florida panthers. Journal of Wildlife Diseases 35:519. DANIELS, M. J., D. BALHARRY, D. HIRST, A. C. KITCHENER, AND R. J. ASPINALL. 1998. Morphological and pelage characteristics of wild living cats in Scotland: implications for defining the wildcat. Journal of Zoology (London) 244: 231-247. ____, M. C. GOLDER, O. JARRETT, AND D. W. MACDONALD. 1999. Feline viruses in wildcats in Scotland. Journal of Wildlife Diseases 35: 121-124. 60

PAGE 72

61 DUNBAR, M. R. 1994. Florida panther biomedical investigation, final performance report. Endangered species project E-1-11 7506, Florida Game and Fresh Water Fish Commission, Tallahassee, Florida, 51 pp. ____. 1995. Florida panther biomedical investigation, annual performance report. Endangered species project E-1-11 7506, Florida Game and Fresh Water Fish Commission, Tallahassee, Florida, 20 pp. ____, P. NOL, AND S. B. LINDA. 1997. Hematologic and serum biochemical reference intervals for Florida panthers. Journal of Wildlife Diseases 33:783-789. CLAUDIA, F., J. L. CATAO-DIAS, G. BAY, E.L. DURIGON, R. S. P. JORGE, C. M. LEUTENEGGER, H. LUTZ, AND R. HOFMANN-LEHMANN. (In press) Journal of Wildlife Diseases. FLYNN, J. N., S. P. DUNHAM, V. WATSON, AND O. JARRETT. 2002. Longitudinal analysis of feline leukemia virus-specific cytotoxic T lymphocytes: correlation with recovery from infection. Journal of Virology 76: 2306-2315. FRANCIS, D. P., M. ESSEX, AND W. D. HARDY. 1977. Excretion of feline leukaemia virus by naturally infected pets. Nature 269: 252-254. ____, ____, AND D. GAYZAGIAN. 1979. Feline-leukemia virus: Survival under home and laboratory conditions. Journal of Clinical Microbiology 9: 154-156. FROMONT, E., M. ARTOIS, M. LANGLAIS, F. COURCHAMP, AND D. PONTIER. 1997. Modeling the feline leukemia virus (FeLV) in natural populations of cats ( Felis catus ). Theoretical Population Biology 52: 60-70. ____, ____, AND D. PONTIER. 1998a. Epidemiology of feline leukemia virus (FeLV) and structure of domestic cat populations. Journal of Wildlife Management 62: 978-988. ____, D. PONTIER, AND M. LANGLAIS. 1998b. Dynamics of a feline retrovirus (FeLV) in host populations with variable spatial structure. Proceedings of the Royal Society of London B 265: 1097-1104. ____, A. SAGER, F. LGER, F. BOURGUEMEISTER, E. JOUQUELET, P. STAHL, D. PONTIER, AND M. ARTOIS. 2000. Prevalence and pathogenicity of retroviruses in wildcats in France. Veterinary Record 146: 317-319. ____, D. PONTIER, AND M. LANGLAIS. 2003. Disease propagation in connected host populations with density-dependent dynamics: the case of the Feline leukemia virus. Journal of Theoretical Biology 223: 465-475. GERSTMAN, B. 1985. The epizootiology of feline leukemia virus infection and its associated diseases. Compendium for Continuing Education 7: 766-774.

PAGE 73

62 GRANT, C. K., M. ESSEX, M. B. GARDNER, W. D. HARDY. 1980. Natural feline leukemia virus infection and the immune response of cats of different ages. Cancer Research 40: 823-829. GRINDEM, C. B., W. T. CORBETT, B. E. AMMERMAN, AND M. T. TOMKINS. 1989. Seroepidemiologic survey of feline immunodeficiency virus infection in cats of Wake County, North Carolina. Journal of the American Veterinary Medical Association 194: 226-228. HARDY, W. D., JR. 1973. Horizontal transmission of feline leukaemia virus. Nature 27: 266-269. ____. 1980a. Feline leukemia virus diseases. In Feline leukemia virus, W. D. HARDY, JR., M. ESSEX, AND A. MCCLELLAND (eds.). Elsevier/North-Holland, New York, New York, pp 3-31. ____. 1980b. The virology, immunology and epidemiology of the feline leukemia virus. In Feline leukemia virus, W. D. HARDY, JR., M. ESSEX, AND A. MCCLELLAND (eds.). Elsevier/North-Holland, New York, New York, pp 33-78. ____, L. J. OLD, P. W. HESS, M. ESSEX, AND S. M. COTTER. 1973. Horizontal transmission of feline leukemia virus. Nature 244: 266-269. ____, P. W. HESS, E. G. MACEWEN, A. J. MCCLELLAND, E. E. ZUCKERMAN, M. ESSEX, S. M. COTTER, AND O. JARRETT. 1976. Biology of feline leukemia virus in the natural environment. Cancer Research 36: 582-588. HARTMANN, K. 2005. Pathogenesis of FeLV. In Clinical advances: A supplement to compendium on continuing education for the practicing veterinarian 27(2A): pp. 8-11. HIETALA, S. K., AND I. A. GARDNER. 1999. Validity of using diagnostic tests that are approved for use in domestic animals for nondomestic species. In Zoo and wild animal medicine, M. E. FOWLER AND R. E. MILLER (eds.). W. B. Saunders Co., Philadelphia, Pennsylvania, pp. 55-58. HOFMANN-LEHMANN, R., E. HOLZNAGEL, A. AUBERT, P. OSSENT, M. REINACHER, AND H. LUTZ. 1995. Recombinant FeLV vaccine: long-term protection and effect on course and outcome of FIV infection. Veterinary Immunology and Immunopathology 46: 127-137. ____, D. FEHR, M. GROB, M. ELGIZOLI, C. PACKER, J. S. MARTENSON, S. J. OBRIEN, H. LUTZ. 1996. Prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in East Africa. Clinical and Diagnostic Laboratory Immunology 3: 554-562.

PAGE 74

63 ____, E. HOLZNAGEL, P. OSSENT, AND H. LUTZ. 1997. Parameters of disease progression in long-term experimental feline retrovirus (feline immunodeficiency virus and feline leukemia virus) infections: hematology, clinical chemistry, and lymphocyte subsets. Clinical and Diagnostic Laboratory Immunology 4: 33-42. ____, J. B. HUDER, S. GRUBER, F. BORETTI, B. SIGRIST, AND H. LUTZ. 2001. Feline leukemia provirus load during the course of experimental infection and in naturally infected cats. Journal of General Virology 82:1589-1596. HOOVER, E. A., AND J. I. MULLINS. 1991. Feline leukemia virus infection and disease. Journal of American Veterinary Medical Association 199: 1287-1297. ____, R. G. OLSEN, W. D. HARDY, JR., J. P. SCHALLER, AND L. E. MATHES. 1976. Feline leukemia virus infection: age-related variation in response of cats to experimental infection. Journal of the National Cancer Institute 57: 365-369. ____, J. L. ROJKO, AND R. G. OLSEN. 1980. Factors influencing host resistance to feline leukemia virus. In Feline leukemia, R. G. OLSEN (ed.). CRC Press, Boca Raton, Florida, pp. 69-76. JACOBSON, R. H., AND N. A. LOPEZ. 1991. Comparative study of diagnostic testing for feline leukemia virus infection. Journal of Veterinary Medical Association 199: 1389-1391. JARRETT, O. 1983. Recent advances in the epidemiology of feline leukaemia virus. Veterinary Annual 23: 287-293. ____, H. M. LAIRD, AND D. HAY. 1973. Determinants of host range of feline leukaemia viruses. Journal of General Virology 20: 169-175. ____, W. D. HARDY, JR., M. C. GOLDER, AND D. HAY. 1978. The frequency of occurrence of feline leukemia virus subgroups in cats. International Journal of Cancer 21: 334-337. ____, M. C. GOLDER, AND K. WEIJER. 1982. A comparison of three methods of feline leukaemia virus diagnosis. The Veterinary Record 110: 325-328. JESSUP, D. A., C. PETTAN, L. J. LOWENSTINE, AND N. C. PEDERSON. 1993. Feline leukemia virus infection and renal spirochetosis in a free-ranging cougar ( Felis concolor ). Journal of Zoo and Wildlife Medicine 24: 73-79. KENNEDY-STOSKOPF, S. 1999. Emerging viral infections in large cats. In Zoo and wild animal medicine, M. E. FOWLER AND R. E. MILLER (eds.). W. B. Saunders Co., Philadelphia, Pennsylvania, pp. 401-410. LAND, E. D., D. R. GARMAN, AND G. A. HOLT. 1998. Monitoring female Florida panthers via cellular telephone. Wildlife Society Bulletin 26: 29-31.

PAGE 75

64 ____, M. CUNNINGHAM, R. MCBRIDE, D. SHINDLE, AND M. LOTZ. 2002. Florida panther genetic restoration and management: annual report. Florida Fish and Wildlife Conservation Commission, Tallahassee, Florida, 111 pp. LEE, I. T., J. K. LEVY, S. P GORMAN, P. C. CRAWFORD, AND M. R. SLATER. 2002. Prevalence of feline leukemia virus infection and serum antibodies against feline immunodeficiency virus in unowned free-roaming cats. Journal of the American Veterinary Medical Association 220:620-622. LEVY, J. K. 1999. FeLV and non-neoplastic FeLV-related disease. In Textbook of veterinary internal medicine, S. J. ETTINGER AND E. C. FELDMAN (eds.). W. B. Saunders Co., Philadelphia, Pennsylvania, pp. 424-432. ____. 2005. Epidemiology, transmissibility, and risk assessment in FeLV. In Clinical advances: A supplement to compendium on continuing education for the practicing veterinarian 27(2A): pp. 4-7. ____, AND C. CRAWFORD. 2005. Feline leukemia virus. In Textbook of veterinary internal medicine, S. J. ETTINGER AND E. C. FELDMAN (eds.). W. B. Saunders Co., Philadelphia, Pennsylvania, pp. 653-659. LOPEZ, N. 1988. Panther study provides new insight into FeLV tests. In Feline health topics pp: 5-8. ____, AND R. H. JACOBSON. 1989. False-positive reactions associated with anti-mouse activity in serotests for feline leukemia virus antigen. Journal of Veterinary Medical Association 195: 741-746. LUBKIN, S. R., J. ROMATOWSKI, M. ZHU, P. M. KULESA, AND K. A. J. WHITE. 1996. Evaluation of feline leukemia virus control measures. Journal of Theoretical Biology 178:53-60. LUTZ, H., N. PEDERSEN, J. HIGGINS, C. W. HARRIS, AND G. H. THEILEN. 1980a. Quantitation of p27 in the serum of cats during natural infection with feline leukemia virus. In Feline leukemia virus, W. D. HARDY, M. ESSEX, AND A. J. MCCLELLAND (eds.). Elsevier/North-Holland, New York, New York, pp. 497-505. ____, ____, ____, U. HUBSCHER, F. A. TROY, AND G. H. THEILEN. 1980b. Humoral immune reactivity to feline leukemia virus and associated antigens in cats naturally infected with feline leukemia virus. Cancer Research 40: 3642-3651. MAEHR, D. S. 1997. The Florida panther: Life and death of a vanishing carnivore. Island Press, Washington, D.C., 261 pp.

PAGE 76

65 MANDEL, M. P., J. R. Stephenson, W. D. Hardy, Jr., and M. Essex. 1979. Endogenous RD-114 virus of cats: absence of antibodies to RD-114 envelope antigens in cats naturally exposed to the feline leukemia virus. Infection and Immunology 24: 282-285. MANSFIELD, K. G., AND E. D. LAND. 2002. Cryptorchidism in Florida panthers: prevalence, features, and influence of genetic variation. Journal of Wildlife Diseases 38: 693-698. MARKER, L., L. MUNSON, P. A. BASSON, AND S. QUACKENBUSH. 2003. Multicentric T-cell lymphoma associated with feline leukemia virus infection in a captive Namibian cheetah ( Acinonyx jubatus ). Journal of Wildlife Diseases 39: 690-695. MCBRIDE, R. 2003. The documented panther population (DPP) and its current distribution from July 1, 2002 to June 30, 2003. Livestock Protection Company, Alpine, Texas, 11 pp. MCCLELLAND, A. J., W. D. HARDY, AND E. E. ZUCKERMAN. 1980. Prognosis of healthy feline leukemia virus infected cats. In Feline leukemia virus, W. D. HARDY, M. ESSEX, AND A. J. MCCLELLAND (eds.). Elsevier/North-Holland, New York, New York, pp. 121-126. MCCOWN, J. W., D. S. MAEHR, AND J. ROBOSKI. 1990. A portable cushion as a wildlife capture aid. Wildlife Society Bulletin 18: 34-36. MCMICHAEL, J. C., S. STIERS, AND S. COFFIN. 1986. Prevalence of feline leukemia virus infection among adult cats at an animal control center: association of viremia with phenotype and season. American Journal of Veterinary Research 47: 765-768. MERIC, S. M. 1984. Suspected feline leukemia virus infection and pancytopenia in a western cougar. Journal of the American Veterinary Medical Association 185: 1390-1391. MIYAZAWA, T. 2002. Infections of feline and feline immunodeficiency virus. Frontiers in Bioscience 7: 504-518. ____, Y. IKEDA, K. MAEDA, T. HORIMOTO, Y. TOHYA, M. MOCHIZUKI, D. VU. G. D. VU, D. X. CU, K. ONO, E. TAKAHASHI, AND T. MIKAMI. 1997. Seroepidemiological survey of feline retrovirus infections in domestic and leopard cats in northern Vietnam in 1997. Journal of Veterinary Medical Science 60: 1273-1275. MOCHIZUKI, M., M. AKUZAWA, AND H. NAGATOMO. 1990. Serological survey of the Iriomote cat ( Felis iriomotensis ) in Japan. Journal of Wildlife Diseases 26: 236-245.

PAGE 77

66 MUNSON, L., L. MARKER, E. DUBOVI, J. A. SPENCER, J. F. EVERMANN, AND S. J. OBRIEN. 2004. Serosurvey of viral infections in free-ranging Namibian cheetahs ( Acinonyx jubatas ). Journal of Wildlife Diseases 40: 23-31. NAKATA, R., T. MIYAZAWA, Y-S. SHIN, R. WATANABE, T. MIKAMI, AND Y. MATSUURA. 2003. Reevaluation of host ranges of feline leukemia virus subgroups. Microbes and Infection 5: 947-950. NEWMAN, A., M. BUSH, D. E. WILDT, D. VAN DAM, M. T. FRANKENHUIS, L. SIMMONS, L. PHILLIPS, AND S. J. OBRIEN. 1985. Biochemical genetic variation in eight endangered or threatened felid species. Journal of Mammalogy 66:256-267. NOWAK, R. M., AND R. MCBRIDE. 1973. Status survey of the Florida panther. Reprinted from the World Wildlife Fund Yearbook, 1973-1974. In Proceedings of the Florida panther conference. Florida Audubon Society and Florida Game and Fresh Water Fish Commission, Orlando, Florida, p. 118. OBRIEN, S. J., M. E. ROELKE, N. YUHKI, K. W. RICHARDS, W. E. JOHNSON, W. L. FRANKLIN, A. E. ANDERSON, O. L. BASS, JR., R. C. BELDEN, AND J. S. MARTENSON. 1990. Genetic introgression within the Florida panther Felis concolor coryi National Geographic Research 6: 485-494. OLMSTEAD, R. A., R. LANGLEY, M. E. ROELKE, R. M. GOEKEN, D. ADGER-JOHNSON, J. P. GOFF, J. P. ALBERT, C. PACKER, M. K. LAURENSON, T. M. CARO, L. SCHEEPERS, D. E. WILDT, M. BUSH, J. S. MARTENSON, AND S. J. OBRIEN. 1992. Worldwide prevalence of lentivirus infection in wild feline species: epidemiologic and phylogenetic aspects. Journal of Virology 66: 6008-6018. OSOFSKY, S. A., K. J. HIRSCH, E. E. ZUCKERMAN, AND W. D. HARDY, JR.. 1996. Feline lentivirus and feline oncovirus status of free-ranging lions ( Panthera leo ), leopards ( Panthera pardus ), and cheetahs ( Acinonyx jubatus ) in Botswana: a regional perspective. Journal of Zoo and Wildlife Medicine 27: 453-467. OSTROWSKI, S., M. VAN VUUREN, D. M. LENAIN, AND A. DURAND. 2003. A serologic survey of wild felids from central west Saudi Arabia. Journal of Wildlife Diseases 39:696-701. PAUL-MURPHY, J., T. WORK, D. HUNTER, E. MCFIE, AND D. FJELLINE. 1994. Serologic survey and serum biochemical reference ranges of the free-ranging mountain lion ( Felis concolor ) in California. Journal of Wildlife Diseases 30: 205-215. PEDERSEN N. C., S. M. MERIC, L. J. JOHNSON, S. P. PLUCKER, AND G. H. THEILEN. 1984. The clinical significance of latent feline leukemia virus infection and cats. Feline Practice 14: 32-48.

PAGE 78

67 ____, M. TORTEN, B. RIDEOUT, E. SPARGER, T. TONACHINI, P. A. LUCIW, C. ACKLEY, N. LEVY, AND J. YAMAMOTO. 1990. Feline leukemia virus infection as a potentiating cofactor for the primary and secondary stages of experimentally induced feline immunodeficiency virus infection. Journal of Virology 64: 598-606. RAMOS-VARA, J. A., M. KIUPEL, AND M. A. MILLER. 2002. Diagnostic immunohistochemistry of infectious diseases in dogs and cats. Journal of Histotechnology 25: 201-214. RASHEED, S., AND M. B. GARDNER. 1981. Isolation of feline leukemia virus from a leopard cat cell line and search for retrovirus in wild felidae. Journal of the National Cancer Institute 67: 929-933. REINACHER, M. 1989. Diseases associated with spontaneous feline leukemia virus (FeLV) infection in cats. Veterinary Immunology and Immunopathology 21: 85-95. ____, G. WITTMER, H. KOBERSTEIN, AND K. FAILING. 1995. Untersuchungen zur bedeutung der FeLVinfektion fur erkrankungen bei sektionskatzen. Berliner und Munchener tierarztliche Wochenschrift. 108: 58-60. RICKARD, L. G., AND W. J. FOREYT. 1992. Gastrointestinal parasites of cougars ( Felis concolor ) in Washington and the first report of Ollulanus tricuspis in a sylvatic felid from North America. Journal of Wildlife Diseases 28: 130133. RIGBY, M. A., J. L. ROJKO, M. A. STEWART, G. J. KOCHIBA, C. M. CHENEY, L. J. REZANKA, L. E. MATHES, J. R. HARTKE, O. JARRETT, AND J. C. NEIL. 1992. Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes. Journal of General Virology 73: 2839-2847. RILEY, S. P. D., J. FOLEY, AND B. CHOMEL. 2004. Exposure to feline and canine pathogens in bobcats and gray foxes in urban and rural zones of a National Park in California. Journal of Wildlife Diseases 40: 11-22. ROELKE, M. E. 1990. Florida panther biomedical investigation, final performance report. Endangered species project E-1-11 7506. Florida Game and Fresh Water Fish Commission, Tallahassee, Florida, 178 pp. ____, J. S. MARTENSON, AND S. J. OBRIEN. 1993a. The consequences of demographic reduction and genetic depletion in the endangered Florida panther. Current Biology 3: 340-350. ____, D. J. FORRESTER, E. R. JACOBSON, G. V. KOLIAS, F. W. SCOTT, M. C. BARR, J. F. EVERMANN, AND E. C. PIRTLE. 1993b. Seroprevalence of infectious disease agents in free-ranging Florida panthers ( Felis concolor coryi ). Journal of Wildlife Diseases 29: 36-49.

PAGE 79

68 ROGERSON, P., W. JARRETT, AND L. MACKEY. 1975. Epidemiological studies on feline leukemia virus infection. International Journal of Cancer 15: 781-785. ROJKO, J. L., AND G. J. KUCIBA. 1991. Pathogenesis of infection by the feline leukemia virus. Journal of the American Veterinary Medical Association 199: 1305-1310. ____, E. A. HOOVER, L. E. MATHES, R. G. OLSEN, AND J. P. SCHALLER. 1979. Pathogenesis of experimental feline leukemia virus infection. Journal of the National Cancer Institute 63: 759-768. ____, ____, S. L. QUACKENBUSH, AND R. G. OLSEN. 1982. Reactivation of latent feline leukaemia virus infection. Nature 298: 385-389. ROTSTEIN, D. S., R. THOMAS, K. HELMICK, S. B. CITINO, S. K., TAYLOR, AND M. R. DUNBAR. 1999. Dermatophyte infections in free-ranging Florida panthers ( Felis concolor coryi ). Journal of Zoo and Wildlife Medicine 30: 281-284. RYSER-DEGIORIS, M. P., R. HOFFMANN-LEHMANN, C. M. LEUTENEGGER, C. HRD AF SEGERSTAD, T. MNER, R. MATTSSON, AND H. LUTZ. 2005. Epizootiologic investigations of selected infectious disease agents in free-ranging Eurasian lynx from Sweden. Journal of Wildlife Diseases 41: 58-66. SARMA, P. S., AND T. LOG. 1973. Subgroup classification of feline leukemia virus and sarcoma virses by viral interference and neutralization tests. Virology 54: 160-169. SCHMITT, A. C., D. REISCHAK, C. L. CAVLAC, C. H. L. MONTFORTE, F. T. COUTO, A. B. P. F. ALMEIDA, D. G. G. SANTOS, L. SOUZA, C. ALVES, K. VECCHI. 2003. Infeco pelos virus da leukemia feline e da peritonite infecciosa feline em felideo selvagem de vida livre e cativeiro da regio do Pantanal matogrossense. Acta Scientiae Veterinariae 31: 185-188. SEAL, U. S. 1994. A plan for genetic restoration and management of the Florida panther ( Felis concolor coryi ). Report to the Florida Game and Fresh Water Fish Commission, Conservation Breeding Specialist Group, SSC/IUCN. White Oak Conservation Center, Yulee, Florida, 23 pp. ____, AND R. LACY. 1989. Florida panther ( Felis concolor coryi ) viability analysis and species survival plan. Report to the U.S. Fish and Wildlife Service. Captive Breeding Specialist Group, SSC/IUCN. Apple Valley, Minnesota, 208 pp. SHINDLE, D., D. LAND, K. CHARLTON, AND R. MCBRIDE. 2000. Florida panther genetic restoration and management: annual report. Florida Fish and Wildlife Conservation Commission, Tallahassee, Florida, 94 pp.

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69 ____, M. CUNNINGHAM, D. LAND, R. MCBRIDE, M. LOTZ, AND B. FERREE. 2003. Florida panther genetic restoration and management: Annual report. Florida Fish and Wildlife Conservation Commission, Tallahassee, Florida, 111 pp. ____, D. LAND, M. W. CUNNINGHAM, M. LOTZ, AND B. FERREE. 2004. Florida panther genetic restoration and management: Annual report. Florida Fish and Wildlife Conservation Commission, Tallahassee, Florida, 102 pp. SLEEMAN, J. M., J. M. KEANE, J. S. JOHNSON, R. J. BROWN, AND S. V. WOUDE. 2001. Feline leukemia virus in a captive bobcat. Journal of Wildlife Diseases 37: 194-200. TAYLOR, S. K., C. D. BUERGELT, M. E. ROELKE-PARKER, B. L. HOMER, AND D. S. ROTSTEIN. 2002. Causes of mortality of free-ranging Florida panthers. Journal of Wildlife Diseases 38: 107-114. TORRES, A. N., C. K. MATHIASON, AND E. A. HOOVER. 2005. Re-examination of feline leukemia virus: host relationships using real-time PCR. Virology 332: 272-283. VANAS, J. 1976. The Florida panther in the Big Cypress Swamp and the role of the Everglades Wonder Gardens in past and future captive breeding programs. In Proceedings of the Florida panther conference. P. C. H. PRICHARD (ed.). Florida Audubon Society and Florida Game and Fresh Water Fish Commission, Orlando, Florida, pp. 109-111.

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BIOGRAPHICAL SKETCH Mark Cunningham was born in Chicago, Illinois, June 16, 1966, and was raised in Miami, Florida. He received his BA in biology from Florida State University in 1991 and graduated from the University of Florida, College of Veterinary Medicine in 1998. He is currently employed as the Division of Wildlife Research veterinarian for the Florida Fish and Wildlife Conservation Commission. 70


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Copyright Date: 2008

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Title: Epizootiology of Feline Leukemia Virus in the Florida Panther
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EPIZOOTIOLOGY OF FELINE LEUKEMIA VIRUS IN THE FLORIDA PANTHER


By

MARK WILLIAM CUNNINGHAM















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Mark William Cunningham

































"The role of disease in wildlife conservation has probably been radically underestimated"
(Aldo Leopold, 1933).















ACKNOWLEDGMENTS

Most of the information presented in this thesis would not be possible without the

expertise of houndsman Roy McBride who began capturing panthers in the 1970s. Also

very much appreciated are the efforts of the biologists on the Florida Fish and Wildlife

Conservation Commission (FWC) panther capture team including Darrell Land, David

Shindle, and Mark Lotz. Veterinarians with FWC who collected samples or data used in

this study include Drs. Melody Roelke, Mike Dunbar, Sharon Taylor, Dave Rotstein, and

Kristin Mansfield. Researchers with the National Park Service also collected samples and

include Deborah Jansen, Steve Schultz, and Dr. Emmett Blankenship.

Veterinary pathologists have been especially helpful with this study and include

Drs. Scott Terrell, Claus Buergelt, and Bruce Homer.

I would especially like to thank my advisor Dr. Donald Forrester for his patience

and guidance. I also appreciate the guidance of other committee members including Drs.

Julie Levy, Mel Sunquist, and Rick Alleman.

I would also like to thank collaborators on this project including Drs. Meredith

Brown, Stephen J. O'Brien, and Warren Johnson at the National Cancer Institute; and

Drs. Kathleen Hayes and Lawrence Mathes at the Ohio State University. I greatly

appreciate the advice and support provided by Drs. Scott Citino, Cynda Crawford, and

William Hardy, Jr. Finally, I am indebted to Richard Kiltie for assistance with statistical

analyses.









This project was fully funded by the Florida Fish and Wildlife Conservation

Commission through the Federal Endangered Species Project E-1 and the Florida Panther

Restoration and Management Trust Fund.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .................................................... ............ ............. .. viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

B background ............................................................... .. ........ ...............
The Florida Panther ................................................. .... ...... .............. ..
Feline Leukemia Virus ................................... ...... ...........4....4
O b j e c tiv e s ........................................................................................................1 1

2 M ATERIALS AND M ETHOD S ........................................ ......................... 13

Study Area and Period .............. .. .......... ...................... ................. 13
Florida Panther Capture and Immobilization .................................. ............... 13
Physical Exam nation ...... ............................... ......... .... .. ................14
Live-capture Sample Collection ...... ..................... .................14
V accination and Treatm ent........................................... ........................... 15
R adio-instrum entation .............. ..................... ................... ............... 16
N e o n ata l K itten s ................................................................................................... 16
N e c ro p sy .......................................................................................................1 6
Specim en Storage ................... .................................................. ...... . ......... 17
Age D term nation and Genetic Status.................................. ....................... 17
D diagnostics ................... .................................................... . ........... 17
Enzyme-linked Immunosorbent Assay Antibody ............................................17
Enzyme-linked Immunosorbent Assay Antigen...............................................17
Immunofluorescent Assay and Immunohistochemistry ....................................18
Polymerase Chain Reaction, Genetic Sequencing, and Viral Culture.................18
Complete Blood Count and Serum Chemistry ..................................................19
O their D iagnostic Testing.......................................................... ............... 19
S ta tistic s .............................................. .. .................... ................ 1 9










3 R E SU L T S ....................................................... 22

D diagnostic T ests...................... .... .................................... ............... ... ............ 22
Enzyme-linked Immunosorbent Assay Antibody ............................................22
Enzyme-linked Immunosorbent Assay Antigen ..............................................23
Immunofluorescent Assay and Immunohistochemistry ....................................24
O th er sero lo g y ................................................... ................ 2 4
C lin ical F in din g s................................................. ................ 2 5
C clinical P ath ology .............................. ......................... ... ...... .. .... ............2 5
P ath o lo g y ...................................................................................................2 5
G r o s s ....................................................................................................... 2 5
M ic ro sc o p ic ............................................................................. 2 6
O opportunistic infections ........................................ .......................... 26
M o reality .................................................................................. 2 6

4 DISCUSSION ....................................................... ........... .... .......... 28

D ia g n o stic s ........................................................................................................... 2 8
E p iz o o tio lo g y ............. ......... .. .... ........... .................................................. 3 0
H history of E xposure.......... ..... .................................................... .. .......... .... 30
Prevalence and D distribution ........................................ .......................... 31
Outcome Following Exposure.................................... ..................... 31
Self-lim iting infections...................... .. .. .......... .. ................. ............... 32
P ersistent infection s.......... ............................................ ........ .... ......... 33
Epizootiology ................................... .......... ......................... 38
C on clu sion ...................... ................ ...................................................... 4 0
Further Research ............... .............. ............... ........ ...... ..............41

APPENDIX

A FLORIDA PANTHER/TEXAS PUMAS SAMPLED DURING THE STUDY
P E R IO D ...................................... ......................................................42

B CASE REPORTS: ANTIGENEMIC FLORIDA PANTHERS..............................49

F P 1 1 5 ....................................................................... 4 9
F P 1 0 9 ....................................................................... 5 1
F P 1 2 2 ....................................................................... 5 2
F P 12 3 ...................................... ......... ..................... ................ 5 4
F P 1 3 2 ....................................................................... 5 5

L IST O F R E F E R E N C E S ......... .................................................................. ............... 60

B IO G R A PH IC A L SK E TCH ..................................................................... ..................70















LIST OF TABLES


Table p

A-i Florida panthers and Texas pumas tested for feline leukemia virus (FeLV)
antigen by ELISA 1 July 2002, to 5 June 2005..................... .............. ............... 43

B-1 Selected hematological and serum biochemical values for Florida panthers
testing positive for feline leukemia virus (FeLV) antigen by ELISA 1 July 2002
to 5 June 2005. ..................................................... ................. 59
















LIST OF FIGURES


Figure p

1-1 Outcome following exposure to feline leukemia virus in domestic cats..................12

2-1 Study area in south Florida, USA ... ..................... .... ..... ................ 21

3-1 Distribution of positive feline leukemia virus positive ELISA antibody optical
densities in Florida panthers/Texas pumas by region and year 1990-2005 ...........23

3-2 Feline leukemia virus (FeLV) ELISA antigen results for panthers >1 yr, not
previously FeLV vaccinated, and sampled in South Forida between 1 July 2002
an d 5 Ju n e 2 0 0 5 .................................................................... 2 7















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EPIZOOTIOLOGY OF FELINE LEUKEMIA VIRUS IN THE FLORIDA PANTHER

By

Mark William Cunningham

August, 2005

Chair: Donald J. Forrester
Major Department: Wildlife Ecology and Conservation

Feline leukemia virus (FeLV) has been reported only rarely in non-domestic felids

and was not detected in Florida panthers (Puma concolor coryi) during almost 20 yr of

routine surveillance. The finding of two FeLV antigen-positive panthers during the 2002-

2003 capture season led to a prospective and retrospective investigation of the

epizootiology of this disease in the population. Archived serum was tested for FeLV

antibodies to assess history of exposure. To determine prevalence and distribution,

panthers were captured throughout their range and tested for FeLV antigen by ELISA.

Positive tests were confirmed by immunofluorescent antibody (IFA) test and viral

culture. The outcome following exposure in panthers was inferred from ELISA antigen

and antibody, IFA, and PCR results. All infected panthers were monitored by radio-

telemetry and necropsied following detection of a mortality signal. Between 1990 and

2005, the prevalence of positive antibody tests increased significantly and were

concentrated in the northern portion of panther range. The prevalence of antigenemia

(positive ELISA antigen) among panthers and Texas pumas >1 yr of age, not previously









vaccinated for FeLV, and sampled between July 2002 and June 2005, was 7% (5 of 71).

Antigenemic panthers were captured or recovered in the Okaloacoochee Slough State

Forest (OKS) in the northern portion of panther range. All antigenemic panthers were

positive by viral culture and three were IFA positive at capture. Clinical signs and clinical

pathology at capture (n = 4) included lymphadenopathy, moderate to severe anemia,

lymphopenia, and acute lymphoblastic leukemia. All infected panthers died during the

study period; causes of deaths were septicemia (n = 2), intraspecific aggression (n = 2),

and unknown (n = 1). Average time from diagnosis to death was 9.25 (SD +10.3) wk in

antigenemic panthers. Following exposure, panthers developed transient, latent, or

persistent infections. The high localized prevalence of antigenemic panthers in OKS

(45.5%) demonstrates the potential impact of this disease on the population. Management

to control the epizootic currently includes vaccination and test-removal. No new cases

have been diagnosed since July 2004.














CHAPTER 1
INTRODUCTION

The Florida panther (Puma concolor coryi) is one of the most endangered mammals

in North America, at one time numbering as few as 30 individuals. With protection and

management the population has rebounded to almost 100; however, the panther is now

threatened by feline leukemia virus (FeLV). Feline leukemia virus infection is a fatal

infectious disease, common to domestic cats (Felis catus), that is rare in non-domestic

felids. Routine FeLV antigen testing in panthers was negative for almost 20 yr until two

positive panthers were detected during the 2002-2003 capture season. These findings

resulted in a prospective and retrospective investigation into the epizootiology of this

disease in the panther population. Information gained from this research will not only be

used to help manage the epizootic in this critically endangered population but may also

benefit managers of other non-domestic felid populations.

Background

The Florida Panther

The Florida panther is an endangered subspecies of puma whose range was once

contiguous with other puma subspecies including the Texas puma (P. concolor

stanlyana). By the early part of the 20th Century; however, habitat destruction,

exploitation, and human population growth had reduced the panther to an isolated

remnant population. The panther was eliminated eventually from all previous range with

the exception of the relatively inaccessible and, historically, undesirable Big Cypress and

Everglades ecosystems of south Florida. Protection of the panther began with state









classification as a game animal in 1950 followed by complete state protection in 1958.

The panther was listed federally as an endangered species in 1967. Nevertheless, the

population dwindled to an estimated 20 to 30 individuals by the early 1970's (Nowak and

McBride, 1973).

Researchers noticed morphologic differences among panthers from different areas

of south Florida. Subsequent genetic analyses revealed two genotypes: 1) original or

canonical Florida panthers, concentrated in the Big Cypress ecosystem, and 2) Florida

panther/South American puma intercrosses which primarily occupied the Everglades

ecosystem (O'Brien et al., 1990). The canonical genotype traced its lineage from the

original remnant population while the South American puma intercrosses likely resulted

from the release of Florida panther/captive puma hybrids into the free-ranging panther

population between 1957 and 1967 (Vanas, 1976). Panthers with genetic evidence of

South American puma ancestry, although representing a minority, had a greater genetic

diversity and fewer congenital anomalies than panthers retaining the canonical genotype

(Roelke et al., 1993a). Among canonical panthers, the level of mitochondrial DNA

variation, frequency of polymorphic allozyme loci, and average heterozygocity of

allozyme loci was lower than any other similarly studied feline except the cheetah

(Acinonyx ubatus) (O'Brien et al., 1990; Newman et al., 1985; Roelke et al., 1993a).

The consequences of inbreeding in panthers were believed to have included

cryptorchidism (Roelke et al., 1993a; Mansfield and Land, 2002), atrial septal defects

(Cunningham et al., 1999), poor seminal traits (Barone et al., 1994), and poor fecundity

(Roelke et al., 1993a). Putative impaired immunocompetence was suspected to increase









susceptibility to parasites and infectious diseases including dermatophytosis (Rotstein et

al., 1999). Many of these traits are still seen in canonical Florida panthers today.

Without intervention the Florida panther was predicted to become extinct within 25

to 40 yr (Seal and Lacy, 1989). However, in 1995 eight female Texas pumas were

released into south Florida as part of a genetic restoration program (Seal, 1994). The

resultant introgression was designed to restore the genetic diversity to levels comparable

to other puma subspecies and to lower the incidence of congenital anomalies in the

panther population.

As of September 2004, over half of the population had Texas puma genes (D. Land,

pers. commun.). The distribution of genotypes was not uniform however, with more

canonical panthers present in the northern portion of panther range. Recent microsatellite

DNA analyses also provided evidence for a third and more recent introgression. Several

captive pumas of unknown western ancestry escaped from the Seminole Indian

Reservation (SIR) north of Big Cypress National Preserve (BCNP) between 1996 and

1999. Although most were eventually recaptured, successful breeding with free-ranging

panthers apparently occurred, and evidence of this genotype was present in 6-10% of

panthers sampled between 2000 and 2004 (D. Land, pers. commun.). This genotype was

concentrated also in the northern portion of panther range.

The prevalence of congenital anomalies among intergrades was reduced greatly and

was limited to the occasional kinked tail or cowlick. As a result of the genetic

introgressions, both deliberate and unintentional, and other management measures, the

panther population had rebounded to a minimum of 87 by 2003 (McBride, 2003).

However, this increase in density may have resulted in an increased risk of infectious









disease transmission and expansion of the wildland-urban interface. These factors may

have set the stage for the current FeLV epizootic.

Feline Leukemia Virus

Feline leukemia virus is a Gammaretrovirus in the family Retroviridae. Following

penetration of the host cell by the viral RNA, reverse transcriptase transcribes viral RNA

into double-stranded DNA which is then incorporated into the host genome. Incorporated

viral DNA, known as provirus, codes for viral proteins and serves as a template for the

production of viral RNA. There are numerous strains of FeLV and few isolates in nature

are identical (Hoover and Mullins, 1991). Feline leukemia virus is classified into

subgroups A, B, and C based on envelope antigens (Jarrett et al., 1973; Sarma and Log;

1973). All FeLV-infected cats carry subgroup A (Jarrett et al., 1978), which is the least

pathogenic and only transmissible form. Subgroup C results from mutation of subgroup

A while subgroup B arises from recombination between subgroup A and endogenous

retroviral DNA (enFeLV) (reviewed by Miyazawa, 2002). EnFeLV are non-coding, non-

immunogenic sequences (Mandel et al., 1979; Rigby et al., 1992) that became

incorporated in the domestic cat genome early in their phylogenetic history. Most non-

domestic felids, including Florida panthers, do not have enFeLV.

The domestic cat is the definitive host for FeLV and the virus has a worldwide

distribution. Although several non-felid cell lines have shown in vitro susceptibility

(Nakata et al., 2003) infection has not been described in non-felid species. The worldwide

prevalence of FeLV in healthy domestic cat populations ranges from 1-8% (Levy, 1999)

with prevalences over 30% in some closed populations (Grant et al., 1980; Gertsmann,

1985). There is evidence that the overall prevalence of FeLV in domestic cats is

decreasing, possibly due to vaccination and other control measures (Levy and Crawford,









2005). In Florida, the prevalence among feral cats is less than 4% (Lee et al., 2002).

Infection is more prevalent among male cats, mixed breed cats, and cats between 1 and 7

yr of age (Levy, 2005). The highest infection rate occurred in cats less than 2 yr of age

(Levy, 1999). In contrast, the prevalence of FeLV antibodies, indicating exposure,

continues to increase with age (Rogerson et al., 1975).

Feline leukemia virus is an enveloped virus and is therefore quite fragile. The virus

immediately begins losing viability outside of the host and, on dry surfaces, is completely

inactivated between two and three hr (Francis et al., 1979). Therefore transmission is

primarily by direct contact. The virus is shed in highest concentrations in the saliva

(Francis et al., 1977), and horizontal transmission occurs primarily via the oronasal route

and by bite wounds. Prolonged contact is generally necessary for effective transmission

(Hardy et al., 1973). Transplacental and transmammary transmission of the virus are also

important (Hardy et al., 1976).

Following exposure most domestic cats will eventually clear the virus while

approximately one-third will become persistently infected and eventually succumb to

FeLV related diseases. However, there is a dynamic relationship between the host and

virus, and progression of disease depends on a number of factors. Outcome following

exposure depends on host age (Hoover et al., 1976), genetics (Hoover and Mullins,

1991), and immunocompetence (Hoover et al., 1980), as well as route of exposure, virus

burden, and strain of virus (Rojko and Kociba, 1991; Hoover and Mullins, 1991). The

progression of infection can be predicted by provirus burden using quantitative

polymerase chain reaction (PCR) (Hofmann-Lehmann et al., 2001). Cats clearing

infection early have no or low provirus burdens, those latently infected retain moderate









levels of provirus, while those becoming persistently infected have high provirus burdens

that peak at about 4 wk post-exposure (Hofmann-Lehmann et al., 2001). These

researchers also demonstrated an inverse correlation between ELISA antibodies and

provirus load beginning approximately 3 wk post-exposure. Cats that resisted persistent

infection had a more pronounced humoral response and lower provirus burdens than cats

that progressed to persistent infections. Cell-mediated immunity is important also in the

early immune response to FeLV infection (Flynn et al., 2002). Regardless of the

outcome, the course of infection is established usually by 8 wk post-exposure (Torres et

al., 2005). Outcome following exposure in domestic cats is summarized in Fig. 1-1.

Following exposure the virus replicates in local lymphoid tissues. Approximately

40% of cats mount an effective immune response and clear the virus before further

progression (Hoover and Mullins, 1991). These cats remain antigen and provirus negative

throughout their lives (Torres et al., 2005). If the infection progresses, however, viral

replication within a small number of circulating leukocytes will lead to infection of

lymphoid organs including the thymus, spleen, and lymph nodes (Rojko et al., 1979).

Cats at this stage may be transiently antigenemic and may even be briefly infectious.

Clinical signs during this primary viremia may include fever, lethargy, leukopenia,

anemia, and lymphadenopathy (Pedersen et al., 1990; Levy, 1999). However,

approximately 50% of cats reaching this stage are still able to mount an effective immune

response and clear the infection (Hoover and Mullins, 1991). Failure of viral containment

will lead to infection of the bone marrow, salivary glands, and other tissues between 3

and 13 wk. Nevertheless, an adequate immune response early in this process may rescue

the cat from persistent infection. These cats will retain provirus in peripheral and marrow









leukocytes for variable periods and are considered latently infected. Latently infected cats

do not shed virus and are not infective to other cats. Reactivation of latent infections

following stress is possible but becomes less likely >1 yr post-infection (Pedersen et al.,

1984). Generally, cats recovering from transient or latent infections are immune to re-

infection.

Progression to persistent infection occurs in approximately 35% of exposed cats

and is characterized by infection of the bone marrow and the development of

cytosuppressive and cytoproliferative diseases. Severity and type of disease in

persistently infected cats depends on host age (Hoover et al., 1976), concurrent feline

immunodeficiency virus (FIV) infection, and virus subgroup and strain. Following

establishment of a persistent infection, a period of dormancy ensues lasting weeks to

years during which few if any clinical signs are apparent. Eventually, persistent infections

result in any of three clinical syndromes: immunosuppression, anemia, and/or neoplasia.

Immunosuppression is believed to result in opportunistic infections. Co-infections

were the most frequent finding in FeLV infected cats examined at North American

veterinary schools (Levy, 1999). Anemia, whether primary or secondary, is the next most

common clinical finding in FeLV infected cats. Anemias are most commonly non-

regenerative and include pure red cell aplasia, red blood cell macrocytosis, erythemic

myelosis, bone marrow infiltration, and anemia of chronic disease (Hardy, 1980a).

Finally, hematopoietic neoplasms may also result from FeLV infection. Lymphoma is the

most common FeLV-related neoplastic disease; leukemias, myeloproliferative diseases,

and fibrosarcomas are also common (Hardy, 1980a). Mortality among persistently









infected cats is approximately 5-fold that of uninfected cats and 83% die within 3.5 yr

(McClelland et al., 1980).

Co-infections of FIV and FeLV are believed to work synergistically to result in

more severe disease (Grindem et al., 1989; Pedersen et al., 1990; Hofmann-Lehmann et

al., 1997). Beebe et al. (1994) suggested that immunosuppression caused by pre-existing

FeLV infection affected disease development upon subsequent FIV infection. Feline

immunodeficiency virus infected cats experimentally infected with FeLV had more

severe disease with a more rapid onset than cats infected with either virus alone. Further,

CD4+ T-lymphocytes were much more depressed in co-infected cats than cats infected

with either virus alone (Hoffmann-Lehmann et al., 1995). It is unknown whether the

order of infection (FeLV or FIV first followed by the other) is important in the clinical

outcome (Hofmann-Lehmann et al., 1997). Finally, virus/virus interactions such as the

formation of FeLV/FIV pseudotypes does not appear to be a mechanism of disease

potentiation (Beebe et al., 1994).

The outcome following introduction of FeLV into naive domestic cat populations

depends on population size, density, dispersal patterns, and spatial and social structure

(Fromont et al., 1998a,b; Fromont et al., 2003). Based on computer models, FeLV

becomes established in large natural domestic cat populations at a prevalence of between

0.8% and 12.4% depending on the parameters used (Fromont et al., 1998a,b) and reduces

population size by 3% (Courchamp et al., 1997) to 7% (Fromont et al., 1997). Inclusion

of FIV in Courchamp's et al. (1997) models more than doubled the population impact of

FeLV. Fromont et al. (1998a) also predicted that FeLV fails to become established in









small isolated populations numbering <100 individuals although extinction of the virus

may take several years.

Feline leukemia virus can be diagnosed and staged using a variety of techniques.

The enzyme-linked immunosorbent assay (ELISA) antigen test is the most common

screening method. The ELISA detects soluble p27 antigen in blood (Lutz et al., 1980a)

usually within 3 wk post-infection (Hofmann-Lehmann et al., 2001). Positive test results

may indicate transient or persistent infection and are an indicator of viremia.

Confirmation of positives is accomplished by immunofluorescent assay (IFA), which

detects p27 antigen within neutrophils and platelets of blood smears (Hardy et al., 1973).

A positive IFA test indicates infection of the bone marrow and usually indicates

persistent infection. Viral culture is highly specific and may be used to detect transient,

latent, or persistent infections and to identify subgroup. Polymerase chain reaction is a

highly sensitive and specific technique that has been used to detect integrated provirus or

free FeLV in formalin-fixed tissues, fresh tissues, bone marrow, and blood. Most

transient and persistent infections are detectable by PCR 1 wk post-infection and all are

detectable by 2 wk (Hofmann-Lehmann et al., 2001). Detection of FeLV antibodies helps

stage the disease, especially identifying previous transient infections, but has little

importance in diagnosis. Feline leukemia virus ELISA antibodies are most frequently

found in those groups clearing the infection (Lutz et al., 1980b). Finally, sequencing of

virus is used to identify strain and subgroup. Expected test results during various stages

of FeLV infection are summarized in Fig. 1-1.

Infection of non-domestic felids by FIV, also a retrovirus, is relatively common and

usually does not result in clinical signs. Approximately 28% of Florida panthers carry the









puma lentivirus strain of FIV (Olmstead et al., 1992) and pathology has not been

observed. In contrast to FIV, FeLV infections in non-domestic felids are quite rare. Feline

leukemia virus infection has been documented in a handful of captive non-domestic felids

including a bobcat (Lynx rufus) (Sleeman et al., 2001), puma (Meric, 1984), clouded

leopard (Neofelis nebulosa) (Citino, 1986), and several cheetahs (A. jubatus) (Briggs and

Ott, 1986; Marker et al., 2003). Feline leukemia virus has also been isolated from a

leopard cat (F. bengalensis) cell line (Rasheed and Gardner, 1981). In all cases, the

source of infection was believed to be infected domestic cats.

Despite extensive testing for FeLV in free-ranging felid populations (Rasheed and

Gardner, 1981; Mochizuki et al., 1990; Roelke et al., 1993b; Paul-Murphy et al., 1994;

Hofmann-Lehmann et al., 1996; Miyazawa et al., 1997; Osofsky et al., 1996; Biek et al.,

2002; Munson et al., 2004; Riley et al., 2004; Ryser-Degioris et al., 2005) published

reports of FeLV infection have been limited to a puma (P. concolor) in California (Jessup

et al., 1993) and a sand cat (F. margarita) in Saudi Arabia (Ostrowski et al., 2003).

Approximately 10 to 24% of European wildcats (F. sylvestris sylvestris) were also FeLV

positive (Daniels et al., 1999; Fromont et al., 2000), although interbreeding with domestic

cats occurs frequently in this subspecies (Daniels et al., 1998).

There have been reports of positive FeLV test results in free-ranging non-domestic

felids that were not confirmed with additional tests. Rickard and Foreyt (1992) detected

FeLV antigen in 2 of 2 free-ranging pumas found dead in Washington but virus isolation

was not attempted. Schmitt et al. (2003) diagnosed FeLV infection by IFA in 11 of 16

(69%) captive and free-ranging felids from Brazil, a biologically inconsistent percentage,

but did not confirm the results by ELISA antigen or viral culture.









Testing for FeLV antibodies has been performed only rarely on samples collected

from non-domestic felids. Feline leukemia virus antibodies were found in a transiently

infected captive clouded leopard (Citino, 1986) and two captive Siberian tigers (Panthera

tigris altaica) (Meric, 1984).

Most infections in non-domestic felids were self-limiting. In a survey of North

American zoos, 7 of 11 (64%) non-domestic felids that originally tested FeLV-positive,

were negative when retested. The remaining four were not retested and did not go on to

develop clinical signs of FeLV. Clinical signs in non-domestic felids with self-limiting

infections were minimal and included lethargy, peripheral lymphadenopathy, and

dehydration. Terminal infections were seen in a free-ranging and captive puma, a bobcat,

and a cheetah. Clinical pathology and necropsy findings included anemia, lymphopenia,

other cytopenias, septicemia, lymphadenopathy, opportunistic infections, and lymphoma

(Meric, 1984; Jessup et al., 1993; Sleeman et al., 2001; Marker et al., 2003).

In Florida panthers, routine FeLV ELISA antigen testing was negative since testing

began in 1978 through late 2002 (Roelke et al., 1993b; Florida Fish and Wildlife

Conservation Commission, unpubl. data); however, during the 2002-2003 capture season,

two panthers tested antigen-positive. These findings launched the investigation detailed

in this report.

Objectives

The objectives of this study were to determine for FeLV in Florida panthers 1) the

history of exposure, 2) the prevalence and geographic distribution, 3) the outcome

following exposure, 4) the clinical signs, clinical pathology, and pathological changes

associated with infection, and 5) risk factors for infection.












INFECTION CLEARED


-65% eventually clear virus


ACTIVE INFECTION

Days



Virerria
Antibody+/-
Antigen +
IFA- 3wks
Rovir + to+++




Virerria
Antibody+/-
Antigen+ 3-13 wks
IFA+
Rovir -++ to ++
Infectias





Persistent Infection
Antibody /-
Antigen Life
IFA + Life


~35% of those exposed will
remain persistently infected


Adaptedfrom: Hartmann(2005), Hoover
and Mullins (1991), and Torres et al. (2005).


Figure 1-1. Outcome following exposure to feline leukemia virus in domestic cats.














CHAPTER 2
MATERIALS AND METHODS

Study Area and Period

Florida panthers were sampled in southern peninsular Florida (south of 280 N)

primarily in the Big Cypress and Everglades ecosystems. For ELISA antibody

comparisons, capture/sampling locations were divided into north and south of 1-75

(approximately 28.050 N) (Fig. 2-1). The prospective study period was 1 July 2002 to 5

June 2005. Archived tissues collected between 1990 and 30 June 2002 were

retrospectively evaluated. For analysis of ELISA antibody prevalence, the study period

was divided into before (1990-1995) and after (1996-2005) genetic restoration.

Previously published and unpublished FeLV ELISA antigen test results from 1983 to 30

June 2002 are included in this report (Roelke, 1990; Roelke et al., 1993b; Dunbar, 1994;

FWC, unpubl. data).

Florida Panther Capture and Immobilization

Free-ranging Florida panthers and translocated Texas cougars were captured using

trained hounds. Panthers either bayed on the ground or were treed, and then were darted

with a 3 ml compressed-air dart fired from a C02-powered rifle. Since 2002,

immobilization drugs included various combinations of ketamine HC1 (Congaree

Veterinary Pharmacy, Cayce, South Carolina, USA), medetomidine (Domitor, Pfizer

Animal Health, Exton, Pennsylvania, USA), tiletamine HCl/zolazepam HC1 (Telazol,

Fort Dodge Animal Health [FDAH], Fort Dodge, Iowa, USA), midazolam HC1 (Abbott

Laboratories, North Chicago, Illinois, USA), and xylazine HC1 (Congaree Veterinary









Pharmacy, USA) (Shindle et al., 2003; Shindle et al., 2004). Following immobilization,

treed panthers were caught with a net and, in some cases, a crash bag (McCown et al.,

1990). Propofol (PropoFloTM, Abbott Laboratories, North Chicago, Illinois, USA) was

administered intravenously (IV) either as a bolus or continuous drip to maintain

anesthesia. Butorphanol tartrate (0.1-0.4 mg/kg, FDAH) or midazolam HC1 (0.03 mg/kg)

was administered intramuscularly (IM) to smooth recovery in some panthers. Panthers

were left to recover in a shaded area away from water. Xylazine HC1 and medetomidine

HC1 were reversed with yohimbine HC1 (Yobine, Lloyd, Inc., Shenandoah, Iowa, USA)

and atipamezol HC1 (Antisedan, Pfizer Animal Health, Exton, Pennsylvania, USA),

respectively, at 12 to 14 their recommended dosages.

Physical Examination

Vital signs (temperature, heart rate, respiration rate, and capillary refill time) and

depth of anesthesia were monitored and recorded. A sterile petrolatum ophthalmic

ointment was applied to the eyes for lubrication. All animals underwent a physical

examination to assess general health and physical condition. For each panther handled,

the skin over the medial saphenous vein was clipped, prepped, and an IV catheter

aseptically placed. Sterile isotonic fluids were administered either subcutaneously (SQ)

or IV. Panthers were implanted with a SQ transponder identification chip (Trovan,

Douglas, United Kingdom), ear-tattooed, measured, and weighed.

Live-capture Sample Collection

Approximately 70-140 ml of blood (depending on body weight) were collected

from the medial saphenous or cephalic veins using a butterfly catheter (19 or 21 gauge),

luer adapter/hub, and Vacutainer tubes (Becton Dickinson, Franklin Lakes, New Jersey,

USA) (approximately 50 ml in serum separator, 40 ml in EDTA, 9 ml in Na Heparin, and









9 ml in ACD tubes). From uncollared panthers, eight skin biopsies (4 mm) were collected

aseptically from the medial aspect of the hindlimbs and saved in biopsy transport media.

Defects were closed with surgical glue. Hair clipped from blood collection and biopsy

sites and pulled hair were saved in sample collection bags; clipped hair was saved also

from the ventral abdomen. Other samples such as bacterial cultures, skin scrapings, and

diagnostic biopsies were taken if indicated. Between November 2002 and April 2004,

blood smears were made from EDTA whole blood on glass slides approximately 6 to 24

hr after collection. Beginning May 2004, blood smears were made in the field from fresh

whole blood.

Vaccination and Treatment

Panthers >4 mo old were vaccinated SQ against feline viral rhinotracheitis (FVR),

feline calicivirus (FCV), feline panleukopenia virus (FPV) (Fel-O-Vax PCT [FDAH], 1

ml, lower left leg), and rabies (RabvacTM 3 [FDAH], 1 ml, lower right leg). Beginning

June 2003, captive and free-ranging panthers were also vaccinated against feline

leukemia virus (FeLV, Fel-O-Vax Lv-K [FDAH] or Fevaxyn FeLV, Schering-Plough

Animal Health Corporation, Omaha, Nebraska, USA, 2 ml, lower left leg). Some

panthers were given a FeLV booster (2 ml) IM remotely by darting 3-16 wk post initial

inoculation. Captured panthers were dewormed with ivermectin (0.1 mg/kg, Ivomec,

Merial Limited, Iselin, New Jersey, USA) and praziquantel (3.75 mg/kg, CestaJectTM

Phoenix Pharmaceutical, Inc., St. Joseph, Missouri, USA) administered SQ in the lateral

aspect of thigh. Penicillin G procaine/benzathine (USVet, Hanford Pharmaceuticals,

Syracuse, New York, USA) was administered IM at 22,000 to 44,000 U/kg.









Radio-instrumentation

Captured adult and juvenile panthers were fitted with a VHF or VHF/GPS radio-

collar and monitored three times weekly as described by Shindle et al. (2004). If a

mortality signal was detected the carcass was recovered the same day for necropsy.

Neonatal Kittens

Neonatal kittens were handled according to Land et al. (1998) and marked with a

SQ transponder identification chip. Pyrantel pamoate (22 mg/kg, Anthelban V, Phoenix

Pharmaceutical, Inc., St. Joseph, Missouri, USA) was administered orally. Blood was

collected from the jugular vein.

Necropsy

All FeLV-positive Florida panthers and/or those found dead due to infectious

disease or unknown causes were completely necropsied by board-certified pathologists at

the University of Florida (Veterinary Medical Teaching Hospital, Gainesville, Florida,

USA) or Disney's Animal Kingdom (Celebration, Florida, USA). One severely autolyzed

FeLV-positive panther (FP109) and all panthers dying of known trauma were necropsied

by the FWC veterinarian at the Wildlife Research Laboratory (FWC, Gainesville, Florida,

USA).

When carcass condition allowed, tissue samples were collected at necropsy from all

major organs. Fluids collected included heart blood, venous blood, thoracic blood,

aqueous humor, and urine. Blood samples were centrifuged at 2000 rpm for 10 minutes

and the supernatant decanted. Representative tissues from fresh (unfrozen) and some

previously frozen panthers were placed in 10% neutral buffered formalin. Fixed tissues

were embedded in paraffin, sectioned at 5 to 6 [tm and stained with hematoxylin and

eosin.









Specimen Storage

All tissues from live-captured and necropsied panthers not immediately analyzed

were archived at -200 to -700C.

Age Determination and Genetic Status

Panther ages were either known (handled as kittens) or were estimated from tooth

wear. Panthers were classified as neonates (<8 wk-old), dependents (8 wk to <1 yr),

subadults (1 to <2.5 yr), adults (2.5 to <10 yr), and older adults (>10 yr).

Panthers were grouped by genotype (canonical Florida panther, Texas puma, Texas

puma/Florida panther intergrade, Texas puma/Everglades/Florida panther intergrade,

SIR/Florida panther intergrade, and Everglades/Florida panther intergrade) (W. Johnson,

unpubl. data).

Diagnostics

Enzyme-linked Immunosorbent Assay Antibody

Antibodies to FeLV were detected at Hansen Veterinary Immunology (Dixon,

California, USA) according to techniques described by Lutz et al. (1980b). Optical

densities (OD) of less than 0.25 were considered negative, 0.25 to <0.35 were low

positive, 0.35 to <0.5 were medium positive, and those >0.500 were high positive. For

statistical analysis any OD >0.25 was considered positive.

Enzyme-linked Immunosorbent Assay Antigen

Serum for ELISA antigen testing (ViraCHEK FeLV, Synbiotics Animal Health,

San Diego, California, USA) was shipped overnight to the New York State Diagnostic

Laboratory (Comell University, Ithaca, New York, USA). Adsorbing reagents were used

to remove heterophile antibody. Fluids collected from live-captured and necropsied

panthers were tested for FeLV antigen with a rapid immunochromatic assay (SNAP









Combo, IDEXX Laboratories, Westbrook, Maine, USA). Beginning November 2003,

EDTA whole blood from captured panthers was tested in the field using the SNAP

combo. The SNAP Combo was also used to test fluids collected from necropsied

panthers. Fluids included blood collected from the thoracic cavity, heart chambers,

vessels, and marrow cavity, and aqueous humor.

Immunofluorescent Assay and Immunohistochemistry

Panthers testing positive by ELISA antigen were also tested by IFA.

Immunofluorescent assays were performed on EDTA or fresh whole blood smears using

techniques described by Hardy et al. (1973) at the National Veterinary Laboratory

(Franklin Lakes, New Jersey, USA). Immunohistochemistry to identify p27 antigen was

performed on formalin-fixed paraffin-embedded tissues at the Diagnostic Center for

Population and Animal Health (Michigan State University, Lansing, Michigan, USA)

using a labeled streptavidin-biotin peroxidase detection system on an automated stainer

(Ramos-Vara et al., 2002).

Polymerase Chain Reaction, Genetic Sequencing, and Viral Culture

Polymerase chain reaction and subsequent genetic sequencing was performed at the

Laboratory for Genomic Diversity (National Cancer Institute, Frederick, Maryland, USA)

on tissues collected from panthers at capture and necropsy. Viral culture was performed

at the Center for Retrovirus Research (The Ohio State University, Columbus, Ohio,

USA). Materials and methods used, and complete results for PCR and genetic sequencing

(M. Brown, unpubl. data) and viral culture (K. Hayes, unpubl. data) will be presented in

separate reports.









Complete Blood Count and Serum Chemistry

Complete blood counts (CBC) and serum biochemical parameters were determined

by Antech Diagnostics (Smyrna, Georgia, USA). Blood smears were examined at the

Veterinary Medical Teaching Hospital (University of Florida, College of Veterinary

Medicine, Gainesville, Florida, USA) for hemoparasites, white blood cell differential

counts, and red blood cell morphology.

Other Diagnostic Testing

Necropsied panthers were tested for rabies by IFA at the Jacksonville Central

Laboratory (Jacksonville, Florida, USA). Viral isolation and real-time and conventional

PCR for canine distemper virus (CDV), pseudorabies virus, flaviviruses, and alphaviruses

were performed at the Southeastern Cooperative Wildlife Disease Study (Athens,

Georgia, USA) on brain, heart, and other tissues collected from panthers dying of

unknown causes.

Other serological tests included Western blot for FIV and kinetics-based enzyme-

linked immunosorbent assay (KELA) for feline coronavirus antibodies (FCV) (New York

State Diagnostic Laboratory). Polymerase chain reaction for Mycoplasma haemofelis and

M. haemominutum was performed on EDTA whole blood from FeLV positive panthers

at the University of Illinois (College of Veterinary Medicine, Urbana, Illinois, USA) and

Cornell University (Ithaca, New York, USA).

Statistics

Prevalence was calculated as the percentage of panthers/pumas positive for FeLV

antibodies by ELISA (OD >0.251). Raw prevalence estimates were examined for each

potential categorical predictor (age classes, genotype, FIV status, location, time period,

and gender). Logistic regression using Egret software (Cytel Software Corporation,









Cambridge, Massachusetts, USA) was performed to investigate ELISA antibody status as

a binary response variable. Odd ratios and their 95% confidence limits were calculated

for each state of the categorical predictors in comparison to an arbitrary reference state.

Significance of difference from 1.0 was determined for the odd ratios by the Wald test.

To account for correlation among replicate outcomes from individuals with multiple test

results, panther identification was modeled as a random effect within the logistic

regression model. Significance of the random effect was evaluated by a likelihood ratio

test. Test results were considered significant at P<0.05. The two significant predictors

emerging from univariate analyses (location and time period) were included in a multiple

predictor logistic regression analysis and their interactions examined.













'U


SF r, .. i:


* Public lands
F[ Private lands


Figure 2-1. Study area in south Florida, USA.


NORTH


SOUTH
SOUTH














CHAPTER 3
RESULTS

Diagnostic Tests

Enzyme-linked Immunosorbent Assay Antibody

ELISA antibody ODs were determined for samples collected from 128 Florida

panthers/Texas pumas on 257 occasions between 2 January 1990 and 29 March 2005.

Eighteen (7%) samples from 17 individuals were positive (1 high OD, 3 medium OD, 14

low OD).

The prevalence of positive antibody ODs was significantly greater in the period

1996-2005 compared to 1990-1995 (P = 0.032). The prevalence of positive antibody ODs

was significantly greater among panthers sampled north of 1-75 compared to south (P =

0.014). No positive ODs were found in the southern portion of panther range (south of

US41). The odds of having a positive antibody OD were not affected by age, gender,

genotype, or FIV status. Of panthers sampled on multiple occasions, six had low or

medium positive ODs at their initial sampling but seroconverted to negative status when

re-sampled 10 mo to 3 yr later.

























0' 1J J W Li North
AWam, CZ LJ. NC
90 91 92 93 94 95 9697 4w Aw 4 W Central
96 9 98 99 O0 I T South
02 03 04 05


Figure 3-1. Distribution of positive feline leukemia virus positive ELISA antibody optical
densities in Florida panthers/Texas pumas by region and year 1990-2005. North
refers to lands north of CR846, NC refers to lands between CR846 and 1-75,
Central refers to lands between 1-75 and US41, and South refers to lands south of
US41.

Enzyme-linked Immunosorbent Assay Antigen

Prior to the study period, all (n = 143 sampled on 322 occasions) Florida panthers

and Texas pumas sampled were negative for p27 antigen by ELISA based on review of

published and unpublished data and retrospective testing. During the study period (1 July

2002 to 5 June 2005), 91 panthers/Texas pumas were tested on 113 occasions for FeLV

antigen by ELISA. Fifty-five panthers or pumas were sampled on 66 occasions at

capture, 40 were sampled at necropsy, 10 were sampled at both capture and necropsy,

and seven were tested as neonatal kittens. Panther number, age, gender, FIV status, and

results of FeLV diagnostic tests are presented in Table A-1.









The prevalence of antigenemia (positive ELISA antigen) among panthers and

Texas pumas >1 yr of age, not previously vaccinated for FeLV, and sampled during the

study period, was 7% (5 of 71). All antigenemic panthers were captured in

Okaloacoochee Slough (OKS). The prevalence of antigenemia in OKS (NC region, Fig.

3-2) was 45.5% (5 of 11).

Antigenemia was only detected in adult panthers (3 males, 2 females). The average

age of antigenemic panthers was 4.85 yr (standard deviation [SD]+3.5) and ranged from

2.25 to 11 yr. Genotypes included canonical Florida panthers (n = 3), Texas puma/Florida

panther intergrade (n = 1), and SIR captive/Florida panther intergrade (n = 1). Case

histories of antigenemic panthers are presented in Appendix B.

Feline leukemia virus antigen was detected by SNAP test in all fluids tested in

those viremic panthers suitable for testing at necropsy. Fluids testing positive included

thoracic blood (FP115, 122, 123, 132), splenic blood (FP115), venous blood (FP132), and

aqueous humor (FP115, 122, 123, 132).

Immunofluorescent Assay and Immunohistochemistry

Three (FP122, 123, 132) of the 5 (60%) panthers positive for FeLV antigen by

ELISA were also IFA positive. Results for two viremic panthers (FP109, 115) were

inconclusive. Spleen and lymph node from 2 of 2 (100%) viremic panthers (FP115, 132)

were positive for p27 antigen by IHC.

Other serology

During the study period, 37.5% of panthers/Texas pumas tested were positive for

FIV antibodies by Western blot. Three of five (60%) FeLV antigen-positive panthers also

tested positive for the puma lentivirus strain of FIV (J. Troyer, unpubl. data). Serology

for FCV was negative for all panthers sampled during the study period (n = 64).









Clinical Findings

Clinical signs observed at capture in four antigenemic panthers included a

peripheral lymphadenopathy (n = 2, 50%) and muscle wasting (n = 1, 25%).

Clinical Pathology

Complete blood counts were performed on four antigenemic panthers sampled

while living. Significant findings included a mild to moderate non-regenerative anemia (n

= 3 [75%]), lymphopenia (n = 3), low hemoglobin (n = 3), monocytosis (n = 1 [25%]),

and elevated nucleated red blood cell count (n = 1). Large immature mononuclear cells

with prominent nucleoli, consistent with acute lymphoblastic leukemia, were seen in two

panthers (FP122, 123, 50%). The mean hematocrit of antigenemic panthers was 29.3%

(SD+ 7.9, range 22.5-42.5%), hemoglobin 9.3 g/dL (SD2.4, range 7.2-13.2 g/dL), red

blood cell count 6.2 x 106/tl (SD+1.78 x 106/tL, range 4.2-8.75 x 106/tl), and

lymphocyte count 1165/4l (SD811.9/4l, range 490-2250/4l). Serum biochemical values

in antigenemic panthers were unremarkable. Clinical pathology of antigenemic panthers

and normal values for panthers are summarized in Table B-1.

Pathology

Gross

Three antigenemic panthers (FP115, 122, 132) were suitable for complete necropsy

based on carcass condition. Completely necropsied panthers had evidence of anemia

(pale mucus membranes and skeletal muscle, n = 2 [66.7%]), moderate to severe

dehydration (n = 2), lymphadenopathy (n = 2), septicemia (n = 2), bronchointerstitial

pneumonia (n = 2), abscesses (n = 1, [33.3%]), and puncture wounds (n = 1). Lacerations

and puncture wounds associated with intraspecific aggression (ISA) were seen in the two

autolyzed/decomposed carcasses (FP109, 123).









Microscopic

Microscopic examination was performed on three panthers (FP115, 122, 132).

Sections of bone marrow from two (66.7%) panthers were hypercellular with

approximately 90 to 100% of the marrow space occupied by hematopoietic cells.

Megakaryocytes were present in normal to moderately increased numbers. No marrow

evidence of acute lymphoblastic leukemia was seen. Microscopic changes consistent with

septicemia were seen in most tissues in FP115 and 132. See Appendix B for complete

histological results.

Opportunistic infections

Aerobic culture of multiple tissues from FP115 and 132 resulted in heavy growth of

Escherichia coli and P-hemolytic Streptococcus sp., respectively. Rabies IFA and viral

isolation and PCR for canine distemper virus, pseudorabies virus, flaviviruses, and

alphaviruses were negative in FP122.

Two of four (50%) ELISA antigen-positive panthers (FP109, 115) were PCR

positive for M. hemominutum; FP115 was also positive for M. haemofelis (J. Messick,

unpubl. data). Organisms were not seen on blood smears made from EDTA whole blood.

Rare Cytauxzoon felis organisms were seen on blood smears from FP109; blood smears

from FP115, 122, and 123 were negative.

Mortality

Suspected causes of death for the five antigenemic panthers included septicemia (n

= 2), intraspecific aggression (n = 2), and anemia/dehydration (n = 1). Time from

diagnosis to death averaged 9.25 (SD+10.3, range 2-24.6) wk in panthers antigenemic at

capture (FP109, 115, 122, 123). Time from diagnosis to death in the two panthers

believed to have died due to FeLV-related diseases was 2 (FP122) and 24.6 wk (FP115).









Time from exposure to death for one panther (FP132) dying of FeLV-related disease was

18.4 wk.


Figure 3-2. Feline leukemia virus (FeLV) ELISA antigen results for panthers >1 yr, not
previously FeLV vaccinated, and sampled in South Forida (south of
Caloosahatchee River) between 1 July 2002 and 5 June 2005.














CHAPTER 4
DISCUSSION

Diagnostics

Diagnostic tests validated for domestic animals but used on wildlife must be

interpreted with caution (Hietala and Gardner, 1999). Nevertheless, the test results in this

study were biologically consistent and appeared to be appropriate and suitable for use in

panthers.

The ELISA antibody test detects exposure to FeLV and is considered more

sensitive but less specific than Western blot analysis. ELISA antibody testing has only

been used rarely in non-domestic felids. Ryser-Degiorgis et al. (2005) found serum from

58 of 102 (58%) Eurasian lynx (L. lynx) to be FeLV positive by ELISA antibody but

negative by Western blot. The authors speculated that cross-reactions with E. coli antigen

(test preparation) or antibodies to murine leukemia viruses may have been responsible for

the false-positive results. Our positive ELISA antibody tests were not confirmed by

Western blot. Further, antibody ODs in panther serum were tested incrementally.

Incremental testing of serum for ELISA antibodies may lead to inconsistent results due to

between-batch variation. Additionally, degradation of antibodies in stored serum may

have resulted in the apparent increase in positive ODs in recently collected samples.

However, FeLV antibodies are stable when frozen in serum (S. Hansen, pers. commun.).

Additionally, ELISA antibody results in Florida panthers were consistent biologically

with other test results and observations. Panthers seroconverted following vaccination

(data not shown) and positive ODs were geographically and temporally clustered.









ELISA antigen tests detect the FeLV p27 protein and therefore should be suitable

for use in exotic species. Nevertheless, false-positives have occurred in some tests that

used murine-derived reagents in domestic and non-domestic cats that had naturally

occurring anti-murine antibodies (Lopez and Jacobson, 1989). False positive results were

reported in one Florida panther tested in 1987 (Lopez, 1988). In this case anti-mouse

antibodies were believed to have resulted from vaccination with a rabies vaccine of

mouse brain origin. Changes in test procedures and reagents effectively eliminated this

problem by the early 1990s (Jacobson and Lopez, 1991). False positives may occur also

due to insufficient washing of vessels in micro-well systems (Jarrett et al., 1982), a

problem not encountered when using rapid immunoassay test kits.

The effectiveness of using body fluids from known infected panthers for detection

of p27 antigen was evaluated. Hemolyzed thoracic, heart, and venous blood; bone

marrow; and aqueous humor from infected panthers consistently tested positive by rapid

immunoassay (SNAP Combo), even on severely autolyzed specimens. The p27 antigen is

only 27,000 daltons and is thus small enough to cross into the aqueous humor in healthy

felids (K. Gellatt, pers. commun.). Thus aqueous humor, and the other fluids described

above, may be useful for FeLV monitoring not only in panthers but in other populations

of pumas.

Immunofluourescent assay and IHC detect p27 antigen in platelets and neutrophils

of blood smears and paraffin-embedded fixed tissues respectively. Three of 5 panthers

positive by ELISA antigen were also positive by IFA; two (FP109, 115) were

inconclusive. Inconclusive results in these panthers may have been due to a delay in

testing and/or improper slide storage. Alternatively, if the samples were true negatives,









sampling may have occurred soon after exposure, before infection of the bone marrow. In

domestic cats neutropenia or thrombocytopenia can lead to false negatives; however,

these values were normal in FP109 and FP115. Spleen and lymph node from FP115 did

test positive by IHC when collected 5 mo after initial positive antigen findings. However,

tissues from FP109 were severely autolyzed and unsuitable for IHC when collected at

necropsy approximately 1 mo after initial positive antigen findings.

Epizootiology


History of Exposure

The FeLV epizootic in free-ranging Florida panthers was foreshadowed by

evidence of increasing exposure based on ELISA antibody tests. Beginning in the late

1990s the prevalence of positive ODs increased dramatically, peaking in 2001 when 9 of

26 (34.6%) were positive (Fig. 3-1). Positive ODs were also geographically clustered

with 16 of 18 (88.9%) positive ODs occurring north of 1-75 (Fig. 3-1). In domestic cats,

antibody ODs increase with age; however, this was not seen in panthers probably the

result of small sample size.

ELISA antibody tests support the theory of multiple introductions of the virus. One

introduction may have occurred on the Florida Panther National Wildlife Refuge

(FPNWR) between January and November of 2001. Four of five (80%) panthers sampled

during the spring of 2001 (2000-2001 capture season) were negative for ELISA

antibodies. Two of these panthers (FP96, 99) were recaptured the next season (2001-

2002). FP96 had seroconverted from a negative to low positive OD, and FP99 went from

a low to medium positive OD. Two other panthers captured in the Fall of 2001 also had

positive ODs (FP107, low; FP78, medium). Based on telemetry data, FP96 and 107









(siblings) and FP99 formed a loosely associated group between August and December

2001 (Land et al., 2002). This may have facilitated exposure among these panthers if any

were shedding virus at the time. Indeed, one of these, FP96, was found to be latently

infected (PCR positive, M. Brown, unpubl. data) at necropsy after being killed by another

male in early 2002. Although no panthers from FPNWR tested antigen-positive when

sampled, we speculate that FP96 became transiently viremic and exposed the panthers

that were accompanying him. He apparently overcame the infection, perhaps aided by a

relatively high antibody OD.

Prevalence and Distribution

Before the 2002-2003 capture season, routine ELISA antigen testing of captured or

necropsied Florida panthers had been negative since 1978. However, between July 2002,

and June 2005, 5 of 71 (8%) free-ranging panthers/pumas >1 yr of age sampled had

active FeLV infections based on ELISA antigen, IFA, and/or viral culture results. All

infected panthers had overlapping home ranges in the OKS ecosystem in the north-central

portion of panther range (Fig. 3-2).

Outcome Following Exposure

In domestic cats, prolonged exposure is generally necessary for transmission.

Indeed the percentage of adult domestic cats becoming persistently infected following a

single exposure event is only 3% (Hartmann, 2005). However, FP132 became infected

apparently after an aggressive encounter with an infected panther (FP123). At

examination approximately 2 days after the fight, FP132 had only minor scratches and

two puncture (presumably bite) wounds. Although FP132 was FeLV antigen negative at

this time, he developed a persistent FeLV infection and died 4 mo later. We speculate

that bite wounds are an important mode of FeLV transmission in panthers. Feline









leukemia virus is present in highest concentrations (106 infectious units/ml) in the saliva

(Francis et al., 1977). A relatively larger dose of saliva would be expected to be

transferred between fighting panthers versus domestic cats and may explain the apparent

ease of transmission.

The presence of infection in females is evidence that transmission also may occur

during breeding. Males and females will pair for 2 to 5 days and transmission may occur

during copulation, mutual grooming, or biting. Although FeLV virus is present in the

semen of domestic cats, venereal transmission is not considered important (Hoover and

Mullins, 1991).

Self-limiting infections

In many respects, the outcome following exposure to the virus in panthers is similar

to that in domestic cats. Following exposure, a panther can clear the virus early

(abortive/transient infections) or can become latently or persistently infected.

Based on the relatively large number of panthers with positive ELISA antibody

ODs but antigen and PCR negative (M. Brown, unpubl. data) test results, many panthers

exposed to the virus are able to clear the infection soon after exposure. Assuming a

similar pathogenesis to that occurring in domestic cats, panthers in this category would

have cleared the infection within several weeks of exposure before infection of the bone

marrow. The majority of domestic cats in this category are considered refractory to re-

infection (Hardy, 1980b). Based on telemetry data, at least one female (FP110) with

evidence of a previous abortive/transient infection survived exposure to at least two

FeLV positive males without developing persistent viremia.

It is possible that FP109 was transiently infected when captured in January 2003.

At capture he was anemic, lymphopenic, and had a profound lymphadenopathy. Levy









(1999) described similar signs in transiently infected domestic cats. FP109 also had a

high ELISA antibody OD. Antibodies detectable by ELISA appear shortly after infection

in domestic cats (Lutz et al., 1980b), and high antibody ODs in domestic cats are a good

prognostic indicator for recovery (Hofmann-Lehmann et al., 2001).

There is also evidence that some panthers can become latently infected as

evidenced by positive PCR and antibody ODs but negative ELISA antigen tests. These

panthers presumably failed to control infection until later in the course of infection, and

retained provirus in leukocytes sufficient to be detectable by PCR. No latently infected

panther has developed a persistent infection, and at least one latently infected panther is

still surviving in the wild at least 2 yr after diagnosis.

Persistent infections

In domestic cats, persistent infection is usually characterized by bone marrow

infection (positive IFA), viremia persisting for >16 wk, and eventual FeLV-related

clinical signs. A diagnosis of persistent infection in panthers was also based on test

results, duration of infection, and clinical signs; however, premature deaths, severe

autolysis, and limited ability to re-sample panthers while living precluded complete

determination of disease progression. Nevertheless, persistent infections were diagnosed

in four panthers (FP115, 122, 123, and 132). These diagnoses were based on viremia >16

wk (FP 115), positive IFA (FP122, 123, 132), and the presence of FeLV-related diseases

(FP115, 122, 123). The latter criterion is speculative; FeLV-infected domestic cats are

subject to the same diseases as non-infected cats (Levy, 1999). Nevertheless, the finding

of septicemia in two necropsied infected panthers and apparent acute lymphoblastic

leukemia in two live-captured infected panthers appears to be unique to those infected









with FeLV. Septicemia without apparent cause or neoplasia was not observed in 73

panther necropsies performed 1978-1999 (Taylor et al., 2002).

Persistently infected panthers had relatively low antibody ODs. Although FP115

had a medium positive OD, FP122, 123, and 132 had negative ODs suggesting a muted

humoral response to infection. In domestic cats, low ELISA antibody ODs are

characteristic of persistent infections (Hoffmann-Lehmann, 2001).

Immunosuppression is a common feature of FeLV infection and is believed to

result in increased susceptibility to infectious diseases. Co-infections were the most

frequent finding in FeLV infected domestic cats examined at North American veterinary

schools (Levy, 1999). Infectious and parasitic diseases seen more commonly in FeLV-

infected domestic cats than non-infected cats included bacterial infections,

hemobartonellosis (Mycoplasma spp.), FCV, upper respiratory infections, babesiosis,

stomatitis, coccidiosis, and toxoplasmosis (Grant et al., 1980; Reinacher, 1989; Reinacher

et al., 1995). Of these, bacterial infections, including p-hemolytic streptococci, were most

important. Jessup et al. (1993) diagnosed septicemia and leptospirosis in a FeLV-infected

puma from California.

The most significant apparent opportunistic infections in panthers were bacterial;

FP 115 had an E. coli septicemia while a mixture of opportunistic bacteria, predominately

0-hemolytic streptococci, was cultured from FP132. Other opportunistic infections in

viremic panthers may have included M. haemofelis and M. haemominutum; however,

approximately 70% of FeLV-negative Florida panthers also tested positive for these

mycoplasmas (J. Messick, unpubl. data). Feline infectious peritonitis has not been

diagnosed in panthers regardless of FeLV status.









Anemias, primarily non-regenerative, are also a frequent finding in FeLV-infected

domestic cats. Non-regenerative anemias were seen in FP109 and FP122 when live

captured, and may have been the cause of death in FP122. Severe anemia in FP132 was

also suspected at necropsy. Bone marrow sections from FP122 and FP132 were examined

histologically. Sections were hypercellular with approximately 90% of the marrow space

occupied by hematopoietic cells; however, erythroid precursors were decreased in

number, and few maturing erythroid cells were present.

Finally, hematopoietic neoplasias occur frequently in domestic cats (Reinacher,

1989). Under controlled conditions opportunistic infections can be reduced and neoplasia

becomes the most important cause of mortality in FeLV-infected cats (Hofmann-

Lehmann et al., 1997). Given the apparently rapid progression of infection, FeLV-

positive panthers may not have survived long enough to develop terminal neoplasia.

Atypical lymphocytes consistent with acute lymphoblastic leukemia were seen on blood

smears from FP122 and FP123; however, no evidence of leukemia was seen on

histological examination of bone marrow or other organ tissue from FP122 (FP123 was

unsuitable for histological examination). The role, if any, in the deaths of these panthers

is unknown.

All antigenemic panthers died relatively soon after diagnosis. FP115 and FP132

died from septicemias (E. coli and P-hemolytic streptococci respectively). FP122 is

believed to have died from severe anemia. FP109 and FP123 died from ISA; however,

anemia or other FeLV-related diseases (acute lymphoblastic leukemia) may have

impaired their ability to fight. For example, FP109 had a hematocrit of 24% (normal is

36.4% [Dunbar et al., 1997]) when handled 1 mo prior to death, which may have resulted









in exercise intolerance. Although FP123 was apparently healthy when handled 6 wk prior

to death, he inflicted only a few minor punctures and scratches to the panther that killed

him (FP132). Further, due to severe autolysis, it is unknown if these panthers may have

died from secondary bacterial infections. Neither FP109 nor FP123 had obviously fatal

ISA-related wounds. Of concern was the presence of healing wounds associated with ISA

in FP132 at the time of death suggesting that he may have exposed another panther

before dying. Further, these bite wounds may have been the source of infection leading to

septicemia in this panther.

Progression of infection appears to be quite rapid in panthers. Although 50% of

viremic domestic cats die within 6 mo of exposure (Jarrett, 1983), adult cats enjoy a

longer induction period and less severe disease compared to younger age groups (Hoover

et al., 1976; Levy, 1999). All viremic panthers were adults and, although the time of

infection is unknown in most infected panthers, the average time from diagnosis to

mortality was just over 9 wk. In the one case of known exposure, FP132 died 18 wk after

exposure to an infected male. Lack of supportive care and presumably increased exposure

to pathogens may play a role in this apparently more rapid clinical course.

Progression to persistent infection following exposure depends on a number of host

and viral factors. The most important host factor in domestic cats is age, but inbreeding

(genetic variation) and FIV status also affect the outcome. Important viral factors include

strain, dose, and duration of exposure. However, with the average age of viremic panthers

approaching 5 yr, maturity did not appear to be protective against infection. Genetic

variation also did not appear to significantly influence the outcome following exposure.

While some panthers had very low genetic variation, at least two had He much greater









than the average for the population (M. Roelke, unpubl. data). Ancestry also did not

appear to play a role. Although three viremic panthers had canonical or original

genotypes, two were intergrades (Florida panther/Texas puma and Florida panther/SIR

captive) (W. Johnson, unpubl. data).

Feline immunodeficiency virus and FeLV, both retroviruses, have overlapping host

cell tropism. In domestic cats, co-infection with FIV results in marked synergism of

immunosuppression and clinical disease induction (Pedersen et al., 1990). Three of five

(60%) FeLV viremic panthers were co-infected with FIV (Shindle et al., 2003; J. Troyer,

unpubl. data); however, the impact of co-infection in the panther is unknown.

Historically, approximately 28% of the free-ranging panther population was

infected with the puma-lentivirus strain of FIV (Olmstead et al., 1992). However, there is

a disturbing trend in FIV prevalence 76% (13 of 17) of panthers captured during the

2004-2005 capture season were positive for FIV antibodies. The consequences of this

trend are unknown but if pre-existing FIV infection affects subsequent FeLV infection,

then an increasing FIV prevalence may alter the epizootiology of FeLV infection in

panthers.

Viral factors may play a more important role in the apparent greater impact of

FeLV on panthers. Although infection pressure is expected to be low in this reclusive

solitary species, viral load may be quite high. Although the virus concentration in saliva

of infected panthers is unknown, the dose transmitted during fighting or breeding would

be expected to be higher as panthers may be as much as 15-fold larger than domestic cats.

However, virus strain may be the most important factor in the current epizootic. Based on









preliminary viral culture results, the strain isolated from the current epizootic in panthers

may be similar to virulent domestic cat strains (K. Hayes, unpubl. data).

Epizootiology

The source of infection in panthers is unknown. Texas pumas introduced in 1995

tested antigen negative during quarantine (Dunbar, 1995), and western pumas in captivity

at SIR tested negative when handled in 1999 (Shindle et al., 2000). In reports of FeLV

infection in non-domestic felids, authors speculated or provided direct evidence that an

infected domestic cat was the source and this is the most likely explanation for FeLV in

panthers. Domestic cat remains have been found in the stomachs of necropsied pumas

from California (Jessup et al., 1993) and there have been observations of panthers killing

domestic cats in Florida (L. Richardson, pers. commun.). Kennedy-Stoskopf (1999)

speculated that consumptionin of FeLV infected domestic cats by larger nondomestic

felids would...be an effective way to transmit the virus."

Private land in panther range continues to be developed at an astounding pace. As

humans encroach on panther habitat they are accompanied by their domestic animals,

including cats. Indeed free-roaming domestic cats have been observed on private lands

near OKS (M. Lotz, pers. commun.). Additionally, the increasing panther population

undoubtedly results in increased opportunities for exposure to domestic cats. Young

dispersing males move through the fringes of the resident population and often occupy

marginal habitat until an established home range becomes available (Maehr, 1997). This

existence on the urban/wildland interface likely increases the risk for exposure to

domestic cats. Riley et al. (2004) speculated that a higher prevalence of positive feline

calicivirus titers in bobcats frequenting urban areas was due to increased exposure to

domestic cats.









The transmission of FeLV from a domestic cat to a panther is likely a rare event.

Domestic cat remains have never been reported in panther scat (Maehr et al., 1990) or in

stomach contents (M. Cunningham, unpubl. data). Further, the odds of a free-ranging

domestic cat in Florida having FeLV is less than 1 in 20 (Lee et al., 2002). Should these

unlikely events occur the panther would still need to become persistently infected

following exposure.

Given that these events, however unlikely, probably did occur, once the species

barrier was crossed the virus was likely spread panther-to-panther. The apparent

transmission of FeLV from FP123 to FP132 supports this theory. Higher panther

densities undoubtedly facilitate this panther-to-panther transmission. The population has

tripled since the early 1990s while panther habitat has been reduced.

Feline leukemia virus infection in panthers is likely a disease of adult cats.

Although mother-to-offspring transmission is probably the most important mode of

transmission in domestic cats (Levy, 2005) it is unlikely to be a factor in the

epizootiology of the disease in panthers. Given the apparent severity and rapid

progression of the disease in panthers, infected females are unlikely to survive to raise

kittens. Further, if a female were to successfully reproduce, infected kittens would be

unlikely to survive to independence the age at which an infected kitten would first be

expected to encounter susceptible panthers and potentially spread the virus.

The current epizootic likely began in the OKS area in late 2001 or early 2002,

possibly as a result of cross-species transmission from an infected domestic cat on a local

ranch. The first positive antibody tests in OKS began to appear in February 2002. The

infection was then likely spread panther-to-panther resulting in the infection of at least









five panthers. However, since July 2004, none of 30 panthers have tested FeLV antigen-

positive indicating that the epizootic may be over. Several factors may have contributed

to this. First, the rapid progression of disease may have limited the number of exposure

events infected panthers die before transmitting the disease. Additionally, some

panthers appear to be refractory to infection thus limiting the number of susceptible

individuals capable of perpetuating the disease. Small population size and geography may

have also helped. Fromont et al. (1998a) demonstrated that populations less than 100

individuals were unlikely to sustain FeLV infections. Since this epizootic occurred in the

northernmost portion of panther range the disease could effectively spread only to the

south. Finally, vaccination may have helped end the epizootic. Vaccination of free-

ranging panthers began in August 2003, and as of June 2005, 34 panthers have received

at least one inoculation. Six of these have died due to non-FeLV causes, therefore, based

on a population size of between 80 and 100, approximately 28 to 35% of the population

has received at least one inoculation. However, because vaccination efforts were targeted

at OKS and adjacent lands, the percentage vaccinated in these areas is much greater.

Using computer models, Lubkin et al. (1996) estimated that 23% to 73% of a population

with a FeLV prevalence of 10% must be effectively vaccinated to eliminate infection.

Conclusion

Kennedy-Stoskopf (1999) speculated "[t]he lack of antigen-positive animals and

absence of clustered clinical cases with FeLV-related diseases are evidence that the virus

is not maintained in [non-domestic felid] populations." However, the finding of five

antigenemic panthers over almost 2 yr is evidence that the disease had, at least

temporarily, become established in the Florida panther population. Small populations are

at greater risk of extinction due to infectious diseases than larger populations









(Berger, 1990). With the exception of the occasional dispersing male, all free-ranging

Florida panthers are part of a single contiguous population in south Florida. As such, the

population is at risk of a catastrophic disease outbreak. Also of concern is the apparent

increased FeLV susceptibility of panthers compared to domestic cats. Transmission

appears to be occurring despite low infection pressure (few exposure events) and host

maturity, and the progression of infection appears to be more rapid compared to domestic

cats. Finally, FeLV prevalence among free-ranging domestic cats in Florida is <4% (Lee

et al., 2002), and in a review of FeLV in domestic cats, Levy (1999) speculated that

"[t]rue 'outbreaks' of FeLV infection are unlikely to occur." However, the prevalence of

FeLV in panthers sampled in OKS went from 0% prior to 2002 to 45.5% between

November 2002, and June 2005. If the disease spreads to the core population the impact

could be devastating.

Further Research

More research is needed to further elucidate the epizootiology of FeLV in panthers.

Western blot antibody tests are needed to confirm positive ELISA antibody tests. Further,

quantitative PCR may be used to estimate provirus burden in panthers. This technique

may be more sensitive than conventional PCR for detecting latent infections. (Hofmann-

Lehmann et al., 2001). In domestic cats latent infections may eventually be cleared.

Testing to determine if living PCR-positive panthers converted to negative status should

be performed to determine the duration of the latent state. Finally, further research is

needed to determine the source of infection. Bobcats should be tested in the OKS region.

Domestic cats from this area should be tested as well, and virus recovered from infected

cats should be sequenced for comparison to strains isolated from panthers.















APPENDIX A
FLORIDA PANTHER/TEXAS PUMAS SAMPLED DURING THE STUDY PERIOD














Table A-1. Florida panthers and Texas pumas tested for feline leukemia virus (FeLV) antigen by ELISA 1 July 2002, to 5 June 2005.
FeLV FIV
Age Antibody Antibody ELISA Western
IDa Date Eventb SexC (yr) Locationd Areae ODf resultg antigenh Blot'


FP 32
FP 48
FP 55
FP 59
FP 59
FP 60
FP 67
FP 69
FP 70
FP 71
FP 73
FP 75
FP 78
FP 79
FP 79
FP 82
FP 83
FP 85
FP 85
FP 86
FP 86
FP 91
FP 91
FP 94
FP 95
FP 98
FP 99
FP 100


9/12/2002
2/18/2005
4/4/2003
4/20/2004
11/22/2004
6/29/2004
1/15/2003
1/3/2005
3/10/2003
2/18/2004
2/27/2003
5/6/2003
10/16/2002
3/17/2004
3/3/2005
12/6/2002
3/31/2004
2/18/2003
3/1/2004
4/1/2003
11/6/2003
3/18/2003
12/12/2003
1/30/2003
3/24/2003
7/1/2002
11/27/2002
1/6/2004


N
L
L
L
N
L
N
L
L
L
L
L
N
L
L
L
L
L
N
L
L
L
N
L
L
N
N
L


15
10.25
10
9.33
9.42
8.75
5.5
7.75
5.83
6.75
6
5
6
8.5
9.5
6
4.8
4
5
3.92
3.92
3.75
4.5
3.5
5.17
4
2.8
7


FPNWR
BCNP-N
BCNP-C
FSSP
PL-Collier Co.
BCNP-S
PL-Collier Co.
BCNP-N
BCNP-C
SIR
BCNP-N
BCNP-N
FPNWR
BCNP-C
BCNP-C
OKS
FSSP
ENP
ENP
BCNP-C
BCNP-C
BCNP-C
BCNP-C
ENP
ENP
SR29
CR846
BCNP-N


North
North
South
South
North
South
North
North
South
North
North
North
North
South
South
North
South
South
South
South
South
South
South
South
South
North
North
North


N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N


0.227
0.188
0.176

0.219

0.252
0.131
0.173
0.17
0.184

0.116
0.176
0.262

0.165

0.165
0.094
0.216

0.141
0.168



0.155














Table A-1. Continued.
FeLV FIV
Age Antibody Antibody ELISA Western
IDa Date Eventb Sex' (yr) Locationd Areae OD result' antigens Bloth


BCNP-C
BCNP-C
BCNP-N
BCNP-C
SR29
FPNWR
BCNP-C
OKS
OKS
OKS
BCNP-N
FPNWR
FPNWR
FPNWR
FPNWR
OKS
OKS
PL-Hendry Co.
PL-Collier Co.
BCNP-N
SIR
OKS
SIR
SIR
BCNP-C
FPNWR


South
South
South
South
North
North
South
North
North
North
North
North
North
North
North
North
North
North
North
North
North
North
North
North
South
North


0.121
0.143
0.132
0.133

0.23

0.546

0.196

0.183
0.237
0.242

0.499

0.149
0.142
0.17
0.157

0.157

0.125
0.237


N N
N N
N N
N N
N
N N
N
H P
S N
N N
N
N N
N N
N N
N
M P
P
N N
N N
N N
N N
N
N N
N
N N
N N


6
3.75
2.13
5.4
3
4.7
1.9
11
11.08
1.08
4
0.54
2
0.54
2.5
4.5


FP 102
FP 103
FP 104
FP 104
FP 106
FP 107
FP 108
FP 109
FP 109
FP 110
FP 112
FP 113
FP 113k
FP 114
FP 114k
FP 115
FP 115
FP 116
FP 116k
FP 117
FP 117
FP 117k
FP 118
FP 118
FP 119
FP 119


N
N
P
P


3/24/2004
2/27/2004
12/13/2002
3/1/2005
2/20/2003
12/6/2004
11/17/2002
1/24/2003
2/21/2003
11/25/2002
9/11/2002
10/23/2002
4/7/2004
10/23/2002
10/17/2003
11/26/2002
5/17/2003
1/20/2003
3/22/2004
2/25/2003
12/3/2003
7/28/2004
3/5/2003
4/2/2003
4/2/2003
11/17/2004


L
L
L
L
N
L
N
L
N
L
N
L
L
L
N
L
N
L
L
L
L
N
L
N
L
L














Table A-1. Continued.
FeLV FIV
Age Antibody Antibody ELISA Western
IDa Date Eventb Sex' (yr) Locationd Areae OD result' antigens Bloth
FP 120 4/8/2003 L F 3 BCNP-C South 0.193 N N N
FP 120 7/14/2004 L F 4 BCNP-C South 0.163 N N P
FP 121 12/2/2003 L F 2.5 SIR North 0.148 N N N
FP 122 1/30/2004 L F 2.25 OKS North 0.183 N P N
FP 122 2/13/2004 N F 2.25 OKS North P -
FP 123 2/2/2004 L M 3.5 OKS North 0.151 N P N
FP 123 3/15/2004 N M 3.6 OKS North P -
FP 124 2/13/2004 L F 3.5 BCNP-S South 0.155 N N P
FP 125 2/13/2004 L M 0.67 BCNP-S South 0.135 N N N
FP 126 2/13/2004 L M 0.67 BCNP-S South 0.155 N N N
FP 126k 5/28/2004 L M 0.96 BCNP-S South 0.163 N N
FP 127 2/16/2004 L M 2 BCNP-C South 0.149 N N P
FP 127k 3/29/2005 L M 3 BCNP-N South 0.196 N N P
FP 128 2/18/2004 L F 3.7 SIR North 0.171 N N N
FP 129 2/20/2004 L F 3 BCNP-C South 0.157 N N P
FP 130 3/4/2004 L M 0.8 OKS North 0.181 N N N
FP 130k 3/10/2005 L M 1.83 PL-Highlands Co. North 0.385 M N P
FP 131 3/10/2004 L M 5 FPNWR North 0.193 N N N
FP 132 3/17/2004 L M 3 OKS North 0.108 N N N
FP 132 7/22/2004 N M 3.33 OKS North 0.23 N P N
FP 133 11/18/2004 L M 4.5 BCNP-N North 0.214 N N P
FP 134 12/14/2004 L M 2.5 BCNP-N North 0.246 N N P
FP 135 12/17/2004 L M 1.7 FPNWR North 0.24 N N N
FP 136 1/13/2005 L F 5 BCNP-C South 0.207 N N N
FP 137 1/25/2005 L M 2.5 OKS North 0.239 N N N
FP 138 1/31/2005 L M 4 BCNP-C South 0.212 N N P
FP 139 3/31/2005 L M 2.83 OKS North 0.213 N N N














Table A-1. Continued.
FeLV FIV
Age Antibody Antibody ELISA Western
IDa Date Eventb Sex' (yr) Locationd Areae OD result' antigens Bloth
K 94 8/17/2004 N M 3 175 North-South N -
K 149 6/11/2003 L F 0.05 OKS North N -
K 150 6/11/2003 L M 0.05 OKS North N -
K 156 8/2/2004 N M 0.5 US41 South N -
K 175 2/10/2005 L M 0.04 BCNP-S South N -
K 176 2/10/2005 L M 0.04 BCNP-S South N -
K 178 3/7/2005 L M 0.05 OKS North N -
K 179 3/7/2005 L F 0.05 OKS North N -
K 180 3/21/2005 L F 0.05 FPNWR North N -
K 181 3/21/2005 L F 0.05 FPNWR North N -
TX 105 1/27/2003 L F 10.5 ENP South 0.161 N N P
TX 106 1/8/2003 L F 9.5 PSSF South 0.129 N N P
TX 108 11/18/2002 L F 10 ENP South 0.189 N N N














Table A-1. Continued.
FeLV FIV
Age Antibody Antibody ELISA Western
IDa Date Eventb Sex' (yr) Locationd Areae OD result' antigens Bloth


CR846
PL
PL
NC
PL
PL
CR858
BCNP
CR833
BCNP-C-S
175
SR29
175
FPNWR
BCNP-C
SR29
SR29
US41
195 Flagler Co.


North
North
North
North
North
North
North
South
North
South
North-South
North
North
North
South
North
North
South
North


0.144 N



0.166 N


UCFP 48
UCFP 49
UCFP 50
UCFP 51
UCFP 54
UCFP 58
UCFP 59
UCFP 60
UCFP 61
UCFP 62
UCFP 63
UCFP 65
UCFP 66
UCFP 67
UCFP 68
UCFP 69
UCFP 70
UCFP 71
UCFP 74


11/11/2002
11/25/2002
1/25/2003
3/10/2003
6/3/2003
6/30/2003
11/2/2003
12/9/2003
12/25/2003
1/11/2004
2/26/2004
4/6/2004
6/27/2004
9/2/2004
9/28/2004
10/25/2004
12/1/2004
2/4/2005
6/4/2005


N
N
N
N
N
N
N
L
N
N
N
N
N
N
N
N
N
N
N


0.7
1.58
6.5
2
0.75
0.83
0.3
1.5
2.5
0.63
3.5
1.5
1.75
0.003
U
2
1
2.5
3


N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N









TABLE A-i LEGEND

aFP (Florida panther), TX (Texas puma), K (Florida panther previously handled as a
neonatal kitten), UCFP (Uncollared Florida panther).

bL (live-capture), N (necropsy)

cF (female), M (male).

dBCNP (Big Cypress National Preserve C [BCNP between 1-75 and US-41], N [BCNP
north of 1-75], S [BCNP south of US-41]), CR (County Road), CWMA (Crew Wildlife
Management Area), ENP (Everglades National Park), FPNWR (Florida Panther National
Wildlife Refuge), I (Interstate), OKS (Okaloacoochee Slough State Forest), PL (private
lands), PSSF (Picayune Strand State Forest), SIR (Big Cypress Seminole Indian
Reservation), SR (State Road), US (United States Road).

eN (panther range north of 1-75), S (panther range south of 1-75), N-S (killed by vehicular
collision on 1-75).
'OD (optical density).

gN (negative), L (low positive), M (medium positive), H (high positive).

hN (negative), P (positive).

'N (negative), P (positive), E (equivocal).

JTest results unsuitable due to severe autolysis.


kPreviously vaccinated for feline leukemia virus.














APPENDIX B
CASE REPORTS: ANTIGENEMIC FLORIDA PANTHERS

FP115

On 26 November 2002, a 4.5 yr-old female Florida panther was captured in OKS.

Capture was routine and the panther appeared healthy weighing 52.7 kg. Physical exam

was unremarkable and routine biomedical samples were collected. The panther was

vaccinated with Fel-O-Vax PCT, de-wormed, radio-instrumented and released.

Complete blood count revealed a mild non-regenerative anemia (28.4%), low

hemoglobin (9.2 g/dL), and lymphopenia (736/4l). Biochemical alterations were limited

to an elevated BUN (57 mg/dL), glucose (183 mg/dL), and creatinine phosphokinase

(609 U/L). FeLV ELISA antigen and FIV ELISA antibody (KELA and Western Blot)

were positive, although IFA of blood smears were inconclusive. Feline leukemia virus

was cultured from EDTA whole blood at the Ohio State University (OSU).

Radio-telemetry over the ensuing 5 mo indicated normal movements with a

minimum convex polygon (MCP) home range of 104.6 km2 (Shindle et al., 2003).

However, between 12 and 17 May 2003 movements became increasingly restricted. On

17 May 2003, the panther was located in a palmetto thicket and died at approximately

1730 hrs.

The panther was immediately collected, placed on ice, and transported to Disney's

Animal Kingdom the next day for complete necropsy. The panther had lost 20.4 kg since

capture; moderate SQ and abdominal fat were present although there was mild muscle

wasting. A copious red-tinged mucosy fluid drained from the nares. The right









submandibular lymph node exuded a bloody purulent yellow fluid. The tracheobronchial

lymph nodes were markedly enlarged and had a nodular appearance on cut section. A

frothy yellow mucoid material was observed at the tracheal bifurcation extending into the

distal airways. The lungs had a diffusely mottled red/black appearance with a nodular

texture which extended into the cut surface. The caudal lung lobes were most severely

affected. The thymus contained multifocal hemorrhages, and the pericardial sac contained

fibrin. The mesenteric lymph nodes were diffusely prominent. The stomach contained a

small amount of hog hair and mucus. The spleen was slightly enlarged and had a meaty

texture. The liver contained 1-3 mm multifocal tan foci. Each ovary contained two 3-5

mm diameter corpora lutea, and the uterus showed no evidence of previous pregnancy.

Histologically, the lungs contained nodular collections of alveoli containing dense

colonies of gram-negative bacteria intermixed with degenerative neutrophils, fibrin,

extravasated erythrocytes, and necrotic debris. There was necrosis of type I pneumocytes

and scattered hyperplasia of type II pneumocytes. Adjacent alveoli contained edema

fluid, macrophages, and neutrophils. Within the kidneys there was multifocal

mineralization of cortical tubules associated with necrosis of tubular epithelial cells. The

tubular epithelial cells contained a golden brown granular pigment. Throughout the

splenic parenchyma there was a mild increase of macrophages with mild hyperplasia of

white pulp. Megakaryocytes were scattered throughout the red pulp. Fibrin was seen in a

few splenic sinuses. Within the submandibular lymph node there was a focally extensive

area of necrosis infiltrated by large numbers of degenerate neutrophils. Other lymph

nodes showed evidence of multifocal cortical hyperplasia with sinuses containing









macrophages and lymphocytes. All other tissues appeared histologically normal.

Immunohistochemistry of spleen and lymph node was positive for p27 antigen.

Aerobic culture of the lung, liver, and submandibular lymph node resulted in pure

growth of E. coli. Viral isolation of the lung was negative.

Although IFA was inconclusive at capture, the persistence of antigenemia for >5

mo combined with clinical signs is consistent with persistent infection.

FP109

FP109 was initially captured and radio-instrumented as a 10 yr-old male 10

February 2002, in OKS. At capture he had injuries consistent with intraspecific

aggression. ELISA ag for FeLV at Cornell University was negative as was ELISA

antibody (HVL). Ten days later FP109 had to be recaptured to replace a defective radio-

collar and ELISA ag was again negative. The radio-collar failed several wk later.

FP109 was recaptured 24 January 2003, at 11 yr of age. At capture he appeared to

be in excellent condition but had a pronounced peripheral lymphadenopathy. Benign

hyperplasia was diagnosed from fine-needle aspirates of the popliteal lymph nodes.

Complete blood count revealed a moderate non-regenerative anemia (23.8%), low

hemoglobin (7.5 g/dL), and lymphopenia (490/4l). Rare C. felis organisms were seen on

blood smears. Serum biochemical abnormalities were minor with only an elevated

glucose (190 mg/dL) and decreased triglycerides (10 mg/dL). Feline leukemia virus

ELISA antigen and antibody tests were positive although IFA of blood smears were

inconclusive. Feline leukemia virus was cultured from EDTA whole blood at OSU.

Radio-telemetry indicated normal movements, but on 27 February 2003, FP109

was found dead. The panther had been dead for 2-3 days, and the carcass was severely

autolyzed, decomposed, and partially scavenged. Partial necropsy revealed puncture









wounds in the skin over the nasal bones with symmetrical crushing fractures of the nasal

bones. Injuries were consistent with ISA.

FP109 died before his true FeLV status could be determined. At capture in January

2003 he was likely in the early stages of infection. Given his high antibody OD, it is

possible he may have eventually cleared the infection. Negative ELISA antigen findings

in heart blood collected at necropsy supports this speculation; however, the sample was

extremely autolyzed and should be considered unreliable.

FP122

On 30 January 2004, a 2.25 yr-old female Florida panther was captured in OKS.

Capture was routine and the panther appeared in poor health weighing only 32.3 kg with

minimal SQ fat. Physical exam was otherwise normal except for a peripheral

lymphadenopathy. Routine biomedical samples were collected, and a SNAP test using

EDTA whole blood in the field was positive. The panther was vaccinated, de-wormed,

radio-instrumented, and released.

Complete blood count revealed a moderate non-regenerative anemia (22.5%), low

hemoglobin (7.2 g/dL), and monocytosis (1020/4l). Mild polychromasia, mild to

moderate anisocytosis, and 10 nucleated red blood cells/100 leukocytes were seen on

peripheral blood smear. Additionally, large immature mononuclear cells that occasionally

contained nucleoli were also seen; these findings were interpreted as an acute

lymphoblastic leukemia. Serum biochemical abnormalities included low cholesterol (62

mg/dL) and triglycerides (9 mg/dL). Abnormalities seen on urinalysis of free-catch urine

included 1+ blood, 3-10 WBC/HPF, 1-3 RBC/HPF, and 4+ bacteria/HPF. Specific

gravity was 1.009. ELISA antigen and IFA were positive. Virus was cultured from EDTA

blood at OSU.









Radio-telemetry over the ensuing 2 wk indicated normal movements and she

remained within the OKS area. Approximately 1 wk after capture, field sign indicated the

panther had killed and fed on a white-tailed deer (Odocoilius virginianus). However, on

13 February 2004, a mortality signal was detected, and her carcass was found in a

hammock in OKS. Time of death was approximately 0500 hrs.

The panther was immediately collected, placed on ice, and transported to Disney's

Animal Kingdom the next day for complete necropsy. At necropsy the panther was

approximately 15% dehydrated, in poor body condition, and had lost 8.9 kg since

capture. Mucus membranes and skeletal muscle were pale. Abdominal and SQ fat were

negligible, and there was evidence of serious fat atrophy. Adrenal glands were diffusely

enlarged. Peripheral lymph nodes were markedly enlarged.

Histologically the bone marrow was hypercellular with approximately 90% of the

marrow space occupied by hematopoietic cells. There was also a moderate increase in the

number of megakaryocytes. Erythroid precursor cells were decreased in number, and few

maturing erythroid cells were present. Myeloid cell lines were relatively increased in

number, and all stages of maturation were observed. Few lymphoid precursors and

mature lymphocytes were seen, and there was no marrow evidence of an acute leukemia.

There was no evidence of cortical follicle formation in examined lymph nodes.

Thymocytes were present in the thymus, but there was no evidence of cortical or

medullary architecture; intermixed among the thymocytes were macrophages containing

a bland golden brown pigment. Alveoli contained eosinophilic fluid and mildly increased

numbers of alveolar macrophages. Within the spleen, much of the red pulp was autolyzed

and there was scattered extramedullary hematopoiesis evident with few megakaryocytes









noted. Within the kidneys, scattered glomeruli were shrunken with markedly thickened

Bowman's capsules, collapse of the glomerular tufts, and replacement of the glomerular

tufts by fibrillar eosinophilic material. Rare cortical tubules were dilated. Scattered few

perivascular infiltrates of lymphocytes and plasma cells were present in the cortical

interstitium.

Rabies IFA was negative. Brain and heart were negative for CDV, pseudorabies

virus, flaviviruses, and alphaviruses by real-time and conventional PCR and viral culture.

Persistent infection was diagnosed based on positive ELISA antigen and IFA

results and clinical signs.

FP123

FP123, a 3.5 yr-old male, was captured 2 February 2004, in OKS. Capture was

routine and the panther appeared healthy weighing approximately 64 kg. Physical exam

was unremarkable and routine biomedical samples were collected. FeLV SNAP test using

EDTA whole blood was positive. The panther was vaccinated, dewormed, radio-

instrumented and released.

Complete blood count abnormalities were limited to a lymphopenia (884/kl),

although a significant percentage (11%) of the differential contained large, apparently

immature, mononuclear cells that occasionally contained nucleoli. These findings were

interpreted as an acute lymphoblastic leukemia. Serum biochemical abnormalities were

suggestive of dehydration and recent feeding (BUN 59 mg/dL, sodium 161 mEq/L,

BUN/creatinine ratio 39, triglycerides 222 mg/dL, and calculated osmolality 336

mOsm/L). FeLV ELISA antigen at Cornell Diagnostic Laboratory and IFA of blood

smears at the National Veterinary Laboratory were positive.









FP123 had a large home range (164.5 km2) traveling at least 10 km to the south

(Shindle et al., 2004). However, within 6 wk of capture, FP123 was found dead 17 March

2004, in OKS following detection of a mortality signal. The carcass was severely

autolyzed and decomposed; date of death was believed to have been 15-16 March. FP132

was captured the same day within 400 m of FP123. Acute injuries on FP132 were

consistent with ISA.

FP123 was completely necropsied at Disney's Animal Kingdom. No gross

abnormalities were noted although the carcass was severely autolysed.

FP132

On 17 March 2004, the carcass of FP123, a FeLV positive male, was recovered in

OKS. External injuries indicated the cause of death to be intraspecific aggression, and the

panther appeared to have been dead for approximately 24-48 hrs. Within 400 m of the

carcass, a freshly killed white-tailed deer was discovered. The dogs were released and

FP132, a 3 yr-old male, was captured. Capture was routine and the panther appeared

healthy weighing 66.3 kg. Two acute puncture wounds over the right shoulder,

presumably bite wounds, were seen on physical examination. Minor lacerations

consistent with claw marks were also seen. Circumstantial evidence was consistent with

FP132 as the cause of death for FP123. Routine biomedical samples were collected and a

SNAP test using EDTA whole blood in the field was negative. The panther was

vaccinated (including 2 ml Fel-O-Vax LvK), de-wormed, radio-instrumented, and

released.

Complete blood count and serum chemistry were unremarkable. Repeat FeLV

ELISA antigen test was negative.









Radio-telemetry over the ensuing 4 mo indicated normal movements with a home

range of approximately 197.4 km2 (Shindle et al., 2004). FP132 was treed and boostered

with 2 ml Fel-O-Vax LvK on 12 April 2004. However, detectable movement based on

radio telemetry ceased between 14 and 21 July 2004. On 20 and 21 July, biologists

investigated and were able to approach to within 5 m of FP132 in thick brush before he

would move ahead. He appeared alert and healthy but lethargic. On 22 July, the panther

was located in a palmetto thicket and appeared to be in respiratory distress. He died at

approximately 1000 hr.

Within 30 min of death, whole blood was collected by dissection of the brachial

artery and aspiration with a needle and syringe. Blood was placed in serum separator and

EDTA tubes, and blood smears were made from cells (EDTA). The panther was

transported from the field and was on ice within 3 hr of death. Necropsy was performed

at Disney's Animal Kingdom. At necropsy the panther was approximately 10%

dehydrated and had lost over 13 kg since capture. There was moderate muscle wasting

although moderate to heavy SQ and abdominal fat was present. A 15x15 mm

pedunculated cutaneous mass was present over the left nasomaxillary region. Mucus

membranes were icteric and pale, and a copious red-tinged fluid drained from the nares.

Several healing puncture wounds and abrasions were noted in multiple sites.

Gross examination revealed a large abscess occupying the subcutis over the lateral

aspect of the right quadriceps muscle. The abscess measured 29x17 cm and had a

variable depth of 5-10 cm. The abscess contained several liters of tan cloudy fluid.

Skeletal muscle was pale. Lungs were diffusely dark red and firmer than expected. On cut

section numerous 1-5 mm tan foci were observed in all lung lobes although the left









cranial and medial lobes were most severely affected. Sections of lung tissue from these

lobes did not float in formalin. The liver was pale and friable. Peripheral lymph nodes

were not significantly enlarged, but mesenteric lymph nodes were larger than expected.

Aerobic cultures were taken of the abscess and lungs resulting in heavy growth of 3-

hemolytic Streptococcus sp.

Histologically, the skeletal muscle beneath the abscess was covered by a thick band

of mixed inflammatory cells representing the margin of the abscess. The superficial

aspect was composed of large numbers of degenerate neutrophils subtended by mixed

macrophages, lymphocytes, and plasma cells as well as immature fibroblasts and

connective tissue markedly expanded by edema. Numerous colonies of large bacterial

cocci were present on the superficial aspect of the lesion. Multifocally throughout the

lung, large dense colonies of bacteria and associated inflammatory cell aggregates

effaced the pulmonary architecture. Smaller bacterial colonies were also common in

airways. Large numbers of degenerate neutrophils and alveolar macrophages were

present in association with bacterial colonies and within the adjacent parenchyma. There

was necrosis of alveolar epithelium with multifocal type II pneumocyte hyperplasia.

Alveoli often contained strands of fibrin and edema fluid. Large areas of necrosis and

hemorrhage were also present. Numerous small (approx 50um diameter) objects

resembling trematode eggs were scattered throughout the liver. These eggs were bounded

by a refractile rim with the central core composed of granular basophilic material.

Numerous eggs were mineralized, degenerate, and were associated with small numbers of

macrophages. Within the thymus there was a loss of architecture and replacement by

abundant adipose tissue. There was also loss of cortico-medullary demarcation with









lymphocytes remaining in a loosely arranged fibrovascular stroma. Scattered cystic

structures were present and were presumed to be Hassals corpuscles. Also present within

the thymus were small mineralized structures which also represented calcified Hassals

corpuscles. Increased numbers of large macrophages containing pale brown cytoplasmic

pigment were present in the thymic parenchyma. Sections of bone marrow were

hypercellular with approximately 100% of the marrow space occupied by hematopoietic

cells. Megakaryocytes were present in normal to mildly increased numbers.

Serum biochemical abnormalities were consistent with hepatic failure (total

bilirubin 5.8 U/L, ALT 455 U/L, and AST 728 U/L) and pre-renal azotemia and/or renal

failure (BUN 63 mg/dL, creatinine 2.8 mg/dL). Other evidence of renal failure included a

severe hyperkalemia (9.7 mEq/L), hyperphosphotemia (19.1 mEq/L), hypermagnesemia,

and calculated osmolality (338 mOsm/L). These findings were likely a combination of

post-mortem artifact (potassium released from platelets), tissue necrosis, metabolic

acidosis, and dehydration. Hypoglycemia (61 mg/dL) was likely the result of septicemia.

FeLV SNAP test of serum and aqueous humor, and IFA of blood smears were

positive. Immunohistochemistry of spleen and lymph node were positive for p27 antigen.

ELISA antigen of serum at Antech Diagnostics was negative, but this is believed to be an

erroneous result. Virus was cultured at OSU.









59






Table B-1. Selected hematological and serum biochemical values for Florida panthers

testing positive for feline leukemia virus (FeLV) antigen by ELISA 1 July

2002 to 5 June 2005.


Panther


Date

Age (yr)

FIVe western blot
FeLV ELISA
FeLV IFA blood smear
Glucose
Blood urea nitrogen
Creatnmne
Total protein
Albumin
Bilrubin
Alkaline phosphatase
Alanlne ammotransferase
Aspartate ammotransferase
Calcium
Phosphorous
Globulin
Creatine phosphoklnase
Hemoglobin
Hematocrit
White blood cells
Red blood cells (RBC)
Mean cell volume
Mean cell hemoglobin (MCH)
MCH concentration
Platelets
Neutrophils
Neutrophils %
Band
Band %
Lymphocytes
Lymphocytes %
Monocytes
Monocytes %
Eoslnophils
Eoslnophils %
Basophils
Basophils %
Nucleated RBC's (NRBC)
T4

Cytauxozoon


FP 109 FP 115 FP 122 FP 123 FP 132

M F F M M

1/24/2003 11/26/2002 1/30/2004 2/2/2004 722/2004
11 45 225 4 3

N P N N N
P P P P P
N N P P
190 183 115 120 61
22 57 24 59 63
20 21 24 15 28
80 66 65 74 55
34 32 33 36 27
01 02 03 03 58
7 12 8 7 4
45 36 35 62 455
24 36 28 68 728
104 93 103 98 102
36 48 50 52 191
46 34 32 38 28
223 609 244 470 223
75 92 72 132 -
23 8 284 225 425
49 92 85 52 -
491 685 418 875
48 41 54 49
153 134 172 151 -
315 324 320 31 1 -
147 236 185 350
4165 7360 4845 3848
85 80 57 74
0 0 0 0
0 0 0 0
490 736 2550 884
10 8 30 17
245 368 1020 416
5 4 12 8
0 736 85 52
0 8 1 1
0 0 0 0
0 0 0 0
0 10 0 -
13 16 13 02 02

P N N N


Units Normalb(SD)C


mg/dl
mg/dl
mg/dl
g/dl
g/dl
mg/dl
U/1
U/1
U/1
mg/dl
mg/dl
g/dl
U/1
g/dl
%


1544(51 0)
377(141)
1 84 (0 54)
7 35 (0 67)
3 70 (0 36)
026(061)
35 4 (386)
60 2 (35 0)
73 4 (77 8)
9 92 (0 66)




515 6 (415 1)
1221 (1 70)


X103/ 1 12 19(3 01)
X106/1l 7635 (1 033)
fl 47 29 (2 89)
Pg 1607(1 41)
g/dl 34 08 (3 26)
X103/1l 4026(131 5)
8000(2900)
% WBC's 643(143)


% WBC's
3400 (1700)
% WBC's 28 8 (145)
390 (340)
% WBC's 3 2 (2 6)
420 (310)
% WBC's 3 4 (2 2)
100 (60)
% WBC's 0 89 (0 57)
'100WBC's 1 5 (1 0)
gg/dl


a ELISA (Enzyme-linked immunosorbent assay).
b Normal values for panthers (Dunbar et al., 1997).
c SD (standard deviation).
d OKS (Okaloacoochee Slough).
SFIV (feline immunodeficiency virus).
fIFA (immunofluorescent assay).















LIST OF REFERENCES


BARONE, M. A., M. E. ROELKE, J. HOWARD, J. L. BROWN, A. E. ANDERSON,
AND D. E. WILDT. 1994. Reproductive characteristics of male Florida panthers:
Comparative studies from Florida, Texas, Colorado, Latin America, and North
American zoos. Journal of Mammalogy 75: 150-162.

BEEBE, A. M., T. G. FAITH, E. E. SPARGER, M. TORTEN, N. C. PEDERSEN, AND
S. DANDEKAR. 1994. Evaluation of in vivo and in vitro interactions of feline
immunodeficiency virus and feline leukemia virus. AIDS 8: 873-878.

BERGER, J. 1990. Persistence of different-sized populations: an empirical assessment
of rapid extinctions in bighorn sheep. Conservation Biology 4: 91-98.

BIEK, R., R. L. ZARNKE, C. GILLIN, M. WILD, J. R. SQUIRES, AND M. POSS.
2002. Serologic survey for viral and bacterial infections in western populations of
Canada lynx (Lynx canadensis). Journal of Wildlife Diseases 38: 840-845.

BRIGGS, M. B., AND R. L. OTT. 1986. Feline leukemia virus infection in a captive
cheetah and the clinical and antibody response of six captive cheetahs to
vaccination with a subunit feline leukemia virus vaccine. Journal of the American
Veterinary Medical Association 189: 1197-1199.

CITINO, S. B. 1986. Transient FeLV viremia in a clouded leopard. Journal of Zoo and
Wildlife Medicine 17: 5-7.

COURCHAMP, F., C. SUPPO, E. FROMONT, AND C. BOULOUX. 1997. Dynamics
of two feline retroviruses (FIV and FeLV) within one population of cats.
Proceedings of the Royal Society of London B 264: 785-794.

CUNNINGHAM, M. W., M. R. DUNBAR, C. D. BUERGELT, B. L. HOMER, S. K.
TAYLOR, R. KING, S. B. CITINO, C. GLASS, AND M. E. ROELKE-PARKER.
1999. Atrial septal defects in Florida panthers. Journal of Wildlife Diseases
35:519-530.

DANIELS, M. J., D. BALHARRY, D. HIRST, A. C. KITCHENER, AND R. J.
ASPINALL. 1998. Morphological and pelage characteristics of wild living cats in
Scotland: implications for defining the 'wildcat'. Journal of Zoology (London) 244:
231-247.

,M. C. GOLDER, 0. JARRETT, AND D. W. MACDONALD. 1999. Feline
viruses in wildcats in Scotland. Journal of Wildlife Diseases 35: 121-124.









DUNBAR, M. R. 1994. Florida panther biomedical investigation, final performance
report. Endangered species project E-l-11 7506, Florida Game and Fresh Water
Fish Commission, Tallahassee, Florida, 51 pp.

S1995. Florida panther biomedical investigation, annual performance report.
Endangered species project E-l-11 7506, Florida Game and Fresh Water Fish
Commission, Tallahassee, Florida, 20 pp.

,P. NOL, AND S. B. LINDA. 1997. Hematologic and serum biochemical
reference intervals for Florida panthers. Journal of Wildlife Diseases 33:783-789.

CLAUDIA, F., J. L. CATAO-DIAS, G. BAY, E.L. DURIGON, R. S. P. JORGE, C. M.
LEUTENEGGER, H. LUTZ, AND R. HOFMANN-LEHMANN. (In press) Journal
of Wildlife Diseases.

FLYNN, J. N., S. P. DUNHAM, V. WATSON, AND O. JARRETT. 2002. Longitudinal
analysis of feline leukemia virus-specific cytotoxic T lymphocytes: correlation with
recovery from infection. Journal of Virology 76: 2306-2315.

FRANCIS, D. P., M. ESSEX, AND W. D. HARDY. 1977. Excretion of feline
leukaemia virus by naturally infected pets. Nature 269: 252-254.

AND D. GAYZAGIAN. 1979. Feline-leukemia virus: Survival under home
and laboratory conditions. Journal of Clinical Microbiology 9: 154-156.

FROMONT, E., M. ARTOIS, M. LANGLAIS, F. COURCHAMP, AND D. PONTIER.
1997. Modeling the feline leukemia virus (FeLV) in natural populations of cats
(Felis catus). Theoretical Population Biology 52: 60-70.

AND D. PONTIER. 1998a. Epidemiology of feline leukemia virus (FeLV)
and structure of domestic cat populations. Journal of Wildlife Management 62:
978-988.

D. PONTIER, AND M. LANGLAIS. 1998b. Dynamics of a feline retrovirus
(FeLV) in host populations with variable spatial structure. Proceedings of the Royal
Society of London B 265: 1097-1104.

A. SAGER, F. LEGER, F. BOURGUEMEISTER, E. JOUQUELET, P. STAHL,
D. PONTIER, AND M. ARTOIS. 2000. Prevalence and pathogenicity of
retroviruses in wildcats in France. Veterinary Record 146: 317-319.

D. PONTIER, AND M. LANGLAIS. 2003. Disease propagation in connected
host populations with density-dependent dynamics: the case of the Feline leukemia
virus. Journal of Theoretical Biology 223: 465-475.

GERSTMAN, B. 1985. The epizootiology of feline leukemia virus infection and its
associated diseases. Compendium for Continuing Education 7: 766-774.









GRANT, C. K., M. ESSEX, M. B. GARDNER, W. D. HARDY. 1980. Natural feline
leukemia virus infection and the immune response of cats of different ages. Cancer
Research 40: 823-829.

GRINDEM, C. B., W. T. CORBETT, B. E. AMMERMAN, AND M. T. TOMKINS.
1989. Seroepidemiologic survey of feline immunodeficiency virus infection in cats
of Wake County, North Carolina. Journal of the American Veterinary Medical
Association 194: 226-228.

HARDY, W. D., JR. 1973. Horizontal transmission of feline leukaemia virus. Nature
27: 266-269.

1980a. Feline leukemia virus diseases. In Feline leukemia virus, W. D. HARDY,
JR., M. ESSEX, AND A. MCCLELLAND (eds.). Elsevier/North-Holland, New
York, New York, pp 3-31.

1980b. The virology, immunology and epidemiology of the feline leukemia virus.
In Feline leukemia virus, W. D. HARDY, JR., M. ESSEX, AND A.
MCCLELLAND (eds.). Elsevier/North-Holland, New York, New York, pp 33-78.

,L. J. OLD, P. W. HESS, M. ESSEX, AND S. M. COTTER. 1973. Horizontal
transmission of feline leukemia virus. Nature 244: 266-269.

,P. W. HESS, E. G. MACEWEN, A. J. MCCLELLAND, E. E. ZUCKERMAN, M.
ESSEX, S. M. COTTER, AND O. JARRETT. 1976. Biology of feline leukemia
virus in the natural environment. Cancer Research 36: 582-588.

HARTMANN, K. 2005. Pathogenesis of FeLV. In Clinical advances: A supplement to
compendium on continuing education for the practicing veterinarian 27(2A): pp. 8-
11.

HIETALA, S. K., AND I. A. GARDNER. 1999. Validity of using diagnostic tests that
are approved for use in domestic animals for nondomestic species. In Zoo and wild
animal medicine, M. E. FOWLER AND R. E. MILLER (eds.). W. B. Saunders
Co., Philadelphia, Pennsylvania, pp. 55-58.

HOFMANN-LEHMANN, R., E. HOLZNAGEL, A. AUBERT, P. OSSENT, M.
REINACHER, AND H. LUTZ. 1995. Recombinant FeLV vaccine: long-term
protection and effect on course and outcome of FIV infection. Veterinary
Immunology and Immunopathology 46: 127-137.

,D. FEHR, M. GROB, M. ELGIZOLI, C. PACKER, J. S. MARTENSON, S. J.
O'BRIEN, H. LUTZ. 1996. Prevalence of antibodies to feline parvovirus,
calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline
leukemia virus antigen and the interrelationship of these viral infections in free-
ranging lions in East Africa. Clinical and Diagnostic Laboratory Immunology 3:
554-562.









E. HOLZNAGEL, P. OSSENT, AND H. LUTZ. 1997. Parameters of disease
progression in long-term experimental feline retrovirus (feline immunodeficiency
virus and feline leukemia virus) infections: hematology, clinical chemistry, and
lymphocyte subsets. Clinical and Diagnostic Laboratory Immunology 4: 33-42.

J. B. HUDER, S. GRUBER, F. BORETTI, B. SIGRIST, AND H. LUTZ. 2001.
Feline leukemia provirus load during the course of experimental infection and in
naturally infected cats. Journal of General Virology 82:1589-1596.

HOOVER, E. A., AND J. I. MULLINS. 1991. Feline leukemia virus infection and
disease. Journal of American Veterinary Medical Association 199: 1287-1297.

R. G. OLSEN, W. D. HARDY, JR., J. P. SCHALLER, AND L. E. MATCHES.
1976. Feline leukemia virus infection: age-related variation in response of cats to
experimental infection. Journal of the National Cancer Institute 57: 365-369.

J. L. ROJKO, AND R. G. OLSEN. 1980. Factors influencing host resistance to
feline leukemia virus. In Feline leukemia, R. G. OLSEN (ed.). CRC Press, Boca
Raton, Florida, pp. 69-76.

JACOBSON, R. H., AND N. A. LOPEZ. 1991. Comparative study of diagnostic testing
for feline leukemia virus infection. Journal of Veterinary Medical Association 199:
1389-1391.

JARRETT, O. 1983. Recent advances in the epidemiology of feline leukaemia virus.
Veterinary Annual 23: 287-293.

H. M. LAIRD, AND D. HAY. 1973. Determinants of host range of feline
leukaemia viruses. Journal of General Virology 20: 169-175.

W. D. HARDY, JR., M. C. GOLDER, AND D. HAY. 1978. The frequency of
occurrence of feline leukemia virus subgroups in cats. International Journal of
Cancer 21: 334-337.

M. C. GOLDER, AND K. WEIJER. 1982. A comparison of three methods of
feline leukaemia virus diagnosis. The Veterinary Record 110: 325-328.

JESSUP, D. A., C. PETTAN, L. J. LOWENSTINE, AND N. C. PEDERSON. 1993.
Feline leukemia virus infection and renal spirochetosis in a free-ranging cougar
(Felis concolor). Journal of Zoo and Wildlife Medicine 24: 73-79.

KENNEDY-STOSKOPF, S. 1999. Emerging viral infections in large cats. In Zoo and
wild animal medicine, M. E. FOWLER AND R. E. MILLER (eds.). W. B.
Saunders Co., Philadelphia, Pennsylvania, pp. 401-410.

LAND, E. D., D. R. GARMAN, AND G. A. HOLT. 1998. Monitoring female Florida
panthers via cellular telephone. Wildlife Society Bulletin 26: 29-31.









,M. CUNNINGHAM, R. MCBRIDE, D. SHINDLE, AND M. LOTZ. 2002.
Florida panther genetic restoration and management: annual report. Florida Fish
and Wildlife Conservation Commission, Tallahassee, Florida, 111 pp.

LEE, I. T., J. K. LEVY, S. P GORMAN, P. C. CRAWFORD, AND M. R. SLATER.
2002. Prevalence of feline leukemia virus infection and serum antibodies against
feline immunodeficiency virus in unowned free-roaming cats. Journal of the
American Veterinary Medical Association 220:620-622.

LEVY, J. K. 1999. FeLV and non-neoplastic FeLV-related disease. In Textbook of
veterinary internal medicine, S. J. ETTINGER AND E. C. FELDMAN (eds.). W.
B. Saunders Co., Philadelphia, Pennsylvania, pp. 424-432.

S2005. Epidemiology, transmissibility, and risk assessment in FeLV. In Clinical
advances: A supplement to compendium on continuing education for the practicing
veterinarian 27(2A): pp. 4-7.

AND C. CRAWFORD. 2005. Feline leukemia virus. In Textbook of veterinary
internal medicine, S. J. ETTINGER AND E. C. FELDMAN (eds.). W. B. Saunders
Co., Philadelphia, Pennsylvania, pp. 653-659.

LOPEZ, N. 1988. Panther study provides new insight into FeLV tests. In Feline health
topics pp: 5-8.

,AND R. H. JACOBSON. 1989. False-positive reactions associated with anti-
mouse activity in serotests for feline leukemia virus antigen. Journal of Veterinary
Medical Association 195: 741-746.

LUBKIN, S. R., J. ROMATOWSKI, M. ZHU, P. M. KULESA, AND K. A. J. WHITE.
1996. Evaluation of feline leukemia virus control measures. Journal of Theoretical
Biology 178:53-60.

LUTZ, H., N. PEDERSEN, J. HIGGINS, C. W. HARRIS, AND G. H. THEILEN.
1980a. Quantitation of p27 in the serum of cats during natural infection with feline
leukemia virus. In Feline leukemia virus, W. D. HARDY, M. ESSEX, AND A. J.
MCCLELLAND (eds.). Elsevier/North-Holland, New York, New York, pp. 497-
505.

U. HUBSCHER, F. A. TROY, AND G. H. THEILEN. 1980b.
Humoral immune reactivity to feline leukemia virus and associated antigens in cats
naturally infected with feline leukemia virus. Cancer Research 40: 3642-3651.

MAEHR, D. S. 1997. The Florida panther: Life and death of a vanishing carnivore.
Island Press, Washington, D.C., 261 pp.









MANDEL, M. P., J. R. Stephenson, W. D. Hardy, Jr., and M. Essex. 1979. Endogenous
RD-114 virus of cats: absence of antibodies to RD-114 envelope antigens in cats
naturally exposed to the feline leukemia virus. Infection and Immunology 24: 282-
285.

MANSFIELD, K. G., AND E. D. LAND. 2002. Cryptorchidism in Florida panthers:
prevalence, features, and influence of genetic variation. Journal of Wildlife
Diseases 38: 693-698.

MARKER, L., L. MUNSON, P. A. BASSON, AND S. QUACKENBUSH. 2003.
Multicentric T-cell lymphoma associated with feline leukemia virus infection in a
captive Namibian cheetah (Acinonyx ubatus). Journal of Wildlife Diseases 39:
690-695.

MCBRIDE, R. 2003. The documented panther population (DPP) and its current
distribution from July 1, 2002 to June 30, 2003. Livestock Protection Company,
Alpine, Texas, 11 pp.

MCCLELLAND, A. J., W. D. HARDY, AND E. E. ZUCKERMAN. 1980. Prognosis of
healthy feline leukemia virus infected cats. In Feline leukemia virus, W. D.
HARDY, M. ESSEX, AND A. J. MCCLELLAND (eds.). Elsevier/North-Holland,
New York, New York, pp. 121-126.

MCCOWN, J. W., D. S. MAEHR, AND J. ROBOSKI. 1990. A portable cushion as a
wildlife capture aid. Wildlife Society Bulletin 18: 34-36.

MCMICHAEL, J. C., S. STIERS, AND S. COFFIN. 1986. Prevalence of feline
leukemia virus infection among adult cats at an animal control center: association
of viremia with phenotype and season. American Journal of Veterinary Research
47: 765-768.

MERIC, S. M. 1984. Suspected feline leukemia virus infection and pancytopenia in a
western cougar. Journal of the American Veterinary Medical Association 185:
1390-1391.

MIYAZAWA, T. 2002. Infections of feline and feline immunodeficiency virus.
Frontiers in Bioscience 7: 504-518.

,Y. IKEDA, K. MAEDA, T. HORIMOTO, Y. TOHYA, M. MOCHIZUKI, D. VU.
G. D. VU, D. X. CU, K. ONO, E. TAKAHASHI, AND T. MIKAMI. 1997.
Seroepidemiological survey of feline retrovirus infections in domestic and leopard
cats in northern Vietnam in 1997. Journal of Veterinary Medical Science 60: 1273-
1275.

MOCHIZUKI, M., M. AKUZAWA, AND H. NAGATOMO. 1990. Serological survey
of the Iriomote cat (Felis iriomotensis) in Japan. Journal of Wildlife Diseases 26:
236-245.









MUNSON, L., L. MARKER, E. DUBOVI, J. A. SPENCER, J. F. EVERMANN, AND S.
J. O'BRIEN. 2004. Serosurvey of viral infections in free-ranging Namibian
cheetahs (Acinonyx jubatas). Journal of Wildlife Diseases 40: 23-31.

NAKATA, R., T. MIYAZAWA, Y-S. SHIN, R. WATANABE, T. MIKAMI, AND Y.
MATSUURA. 2003. Reevaluation of host ranges of feline leukemia virus
subgroups. Microbes and Infection 5: 947-950.

NEWMAN, A., M. BUSH, D. E. WILDT, D. VAN DAM, M. T. FRANKENHUIS, L.
SIMMONS, L. PHILLIPS, AND S. J. O'BRIEN. 1985. Biochemical genetic
variation in eight endangered or threatened felid species. Journal of Mammalogy
66:256-267.

NOWAK, R. M., AND R. MCBRIDE. 1973. Status survey of the Florida panther.
Reprinted from the World Wildlife Fund Yearbook, 1973-1974. In Proceedings of
the Florida panther conference. Florida Audubon Society and Florida Game and
Fresh Water Fish Commission, Orlando, Florida, p. 118.

O'BRIEN, S. J., M. E. ROELKE, N. YUHKI, K. W. RICHARDS, W. E. JOHNSON, W.
L. FRANKLIN, A. E. ANDERSON, O. L. BASS, JR., R. C. BELDEN, AND J. S.
MARTENSON. 1990. Genetic introgression within the Florida panther Felis
concolor coryi. National Geographic Research 6: 485-494.

OLMSTEAD, R. A., R. LANGLEY, M. E. ROELKE, R. M. GOEKEN, D. ADGER-
JOHNSON, J. P. GOFF, J. P. ALBERT, C. PACKER, M. K. LAURENSON, T. M.
CARO, L. SCHEEPERS, D. E. WILDT, M. BUSH, J. S. MARTENSON, AND S.
J. O'BRIEN. 1992. Worldwide prevalence of lentivirus infection in wild feline
species: epidemiologic and phylogenetic aspects. Journal of Virology 66: 6008-
6018.

OSOFSKY, S. A., K. J. HIRSCH, E. E. ZUCKERMAN, AND W. D. HARDY, JR..
1996. Feline lentivirus and feline oncovirus status of free-ranging lions (Panthera
leo), leopards (Panthera pardus), and cheetahs (Acinonyx jubatus) in Botswana: a
regional perspective. Journal of Zoo and Wildlife Medicine 27: 453-467.

OSTROWSKI, S., M. VAN VUUREN, D. M. LENAIN, AND A. DURAND. 2003. A
serologic survey of wild felids from central west Saudi Arabia. Journal of Wildlife
Diseases 39:696-701.

PAUL-MURPHY, J., T. WORK, D. HUNTER, E. MCFIE, AND D. FJELLINE. 1994.
Serologic survey and serum biochemical reference ranges of the free-ranging
mountain lion (Felis concolor) in California. Journal of Wildlife Diseases 30: 205-
215.

PEDERSEN N. C., S. M. MERIC, L. J. JOHNSON, S. P. PLUCKER, AND G. H.
THEILEN. 1984. The clinical significance of latent feline leukemia virus infection
and cats. Feline Practice 14: 32-48.









,M. TORTEN, B. RIDEOUT, E. SPARGER, T. TONACHINI, P. A. LUCIW, C.
ACKLEY, N. LEVY, AND J. YAMAMOTO. 1990. Feline leukemia virus
infection as a potentiating cofactor for the primary and secondary stages of
experimentally induced feline immunodeficiency virus infection. Journal of
Virology 64: 598-606.

RAMOS-VARA, J. A., M. KIUPEL, AND M. A. MILLER. 2002. Diagnostic
immunohistochemistry of infectious diseases in dogs and cats. Journal of
Histotechnology 25: 201-214.

RASHEED, S., AND M. B. GARDNER. 1981. Isolation of feline leukemia virus from a
leopard cat cell line and search for retrovirus in wild felidae. Journal of the
National Cancer Institute 67: 929-933.

REINACHER, M. 1989. Diseases associated with spontaneous feline leukemia virus
(FeLV) infection in cats. Veterinary Immunology and Immunopathology 21: 85-95.

,G. WITTMER, H. KOBERSTEIN, AND K. FAILING. 1995. Untersuchungen
zur bedeutung der FeLV- infection fur erkrankungen bei sektionskatzen. Berliner
und Munchener tierarztliche Wochenschrift. 108: 58-60.

RICKARD, L. G., AND W. J. FOREYT. 1992. Gastrointestinal parasites of cougars
(Felis concolor) in Washington and the first report of Ollulanus tricuspis in a
sylvatic felid from North America. Journal of Wildlife Diseases 28: 130133.

RIGBY, M. A., J. L. ROJKO, M. A. STEWART, G. J. KOCHIBA, C. M. CHENEY, L.
J. REZANKA, L. E. MATCHES, J. R. HARTKE, O. JARRETT, AND J. C. NEIL.
1992. Partial dissociation of subgroup C phenotype and in vivo behaviour in feline
leukaemia viruses with chimeric envelope genes. Journal of General Virology 73:
2839-2847.

RILEY, S. P. D., J. FOLEY, AND B. CHOMEL. 2004. Exposure to feline and canine
pathogens in bobcats and gray foxes in urban and rural zones of a National Park in
California. Journal of Wildlife Diseases 40: 11-22.

ROELKE, M. E. 1990. Florida panther biomedical investigation, final performance
report. Endangered species project E-l-11 7506. Florida Game and Fresh Water
Fish Commission, Tallahassee, Florida, 178 pp.

,J. S. MARTENSON, AND S. J. O'BRIEN. 1993a. The consequences of
demographic reduction and genetic depletion in the endangered Florida panther.
Current Biology 3: 340-350.

,D. J. FORRESTER, E. R. JACOBSON, G. V. KOLIAS, F. W. SCOTT, M. C.
BARR, J. F. EVERMANN, AND E. C. PIRTLE. 1993b. Seroprevalence of
infectious disease agents in free-ranging Florida panthers (Felis concolor coryi).
Journal of Wildlife Diseases 29: 36-49.









ROGERSON, P., W. JARRETT, AND L. MACKEY. 1975. Epidemiological studies on
feline leukemia virus infection. International Journal of Cancer 15: 781-785.

ROJKO, J. L., AND G. J. KUCIBA. 1991. Pathogenesis of infection by the feline
leukemia virus. Journal of the American Veterinary Medical Association 199:
1305-1310.

,E. A. HOOVER, L. E. MATCHES, R. G. OLSEN, AND J. P. SCHALLER. 1979.
Pathogenesis of experimental feline leukemia virus infection. Journal of the
National Cancer Institute 63: 759-768.

S. L. QUACKENBUSH, AND R. G. OLSEN. 1982. Reactivation of latent
feline leukaemia virus infection. Nature 298: 385-389.

ROTSTEIN, D. S., R. THOMAS, K. HELMICK, S. B. CITINO, S. K., TAYLOR, AND
M. R. DUNBAR. 1999. Dermatophyte infections in free-ranging Florida panthers
(Felis concolor coryi). Journal of Zoo and Wildlife Medicine 30: 281-284.

RYSER-DEGIORIS, M. P., R. HOFFMANN-LEHMANN, C. M. LEUTENEGGER, C.
HARD AF SEGERSTAD, T. MONER, R. MATTSSON, AND H. LUTZ. 2005.
Epizootiologic investigations of selected infectious disease agents in free-ranging
Eurasian lynx from Sweden. Journal of Wildlife Diseases 41: 58-66.

SARMA, P. S., AND T. LOG. 1973. Subgroup classification of feline leukemia virus
and sarcoma virses by viral interference and neutralization tests. Virology 54: 160-
169.

SCHMITT, A. C., D. REISCHAK, C. L. CAVLAC, C. H. L. MONTFORTE, F. T.
COUTO, A. B. P. F. ALMEIDA, D. G. G. SANTOS, L. SOUZA, C. ALVES, K.
VECCHI. 2003. Infegcco pelos virus da leukemia feline e da peritonite infecciosa
feline em felideo selvagem de vida livre e cativeiro da regido do Pantanal
matogrossense. Acta Scientiae Veterinariae 31: 185-188.

SEAL, U. S. 1994. A plan for genetic restoration and management of the Florida
panther (Felis concolor coryi). Report to the Florida Game and Fresh Water Fish
Commission, Conservation Breeding Specialist Group, SSC/IUCN. White Oak
Conservation Center, Yulee, Florida, 23 pp.

,AND R. LACY. 1989. Florida panther (Felis concolor coryi) viability analysis
and species survival plan. Report to the U.S. Fish and Wildlife Service. Captive
Breeding Specialist Group, SSC/IUCN. Apple Valley, Minnesota, 208 pp.

SHINDLE, D., D. LAND, K. CHARLTON, AND R. MCBRIDE. 2000. Florida panther
genetic restoration and management: annual report. Florida Fish and Wildlife
Conservation Commission, Tallahassee, Florida, 94 pp.









M. CUNNINGHAM, D. LAND, R. MCBRIDE, M. LOTZ, AND B. FERREE.
2003. Florida panther genetic restoration and management: Annual report. Florida
Fish and Wildlife Conservation Commission, Tallahassee, Florida, 111 pp.

D. LAND, M. W. CUNNINGHAM, M. LOTZ, AND B. FERREE. 2004. Florida
panther genetic restoration and management: Annual report. Florida Fish and
Wildlife Conservation Commission, Tallahassee, Florida, 102 pp.

SLEEMAN, J. M., J. M. KEANE, J. S. JOHNSON, R. J. BROWN, AND S. V. WOUDE.
2001. Feline leukemia virus in a captive bobcat. Journal of Wildlife Diseases 37:
194-200.

TAYLOR, S. K., C. D. BUERGELT, M. E. ROELKE-PARKER, B. L. HOMER, AND
D. S. ROTSTEIN. 2002. Causes of mortality of free-ranging Florida panthers.
Journal of Wildlife Diseases 38: 107-114.

TORRES, A. N., C. K. MATHIASON, AND E. A. HOOVER. 2005. Re-examination of
feline leukemia virus: host relationships using real-time PCR. Virology 332: 272-
283.

VANAS, J. 1976. The Florida panther in the Big Cypress Swamp and the role of the
Everglades Wonder Gardens in past and future captive breeding programs. In
Proceedings of the Florida panther conference. P. C. H. PRICHARD (ed.). Florida
Audubon Society and Florida Game and Fresh Water Fish Commission, Orlando,
Florida, pp. 109-111.















BIOGRAPHICAL SKETCH

Mark Cunningham was born in Chicago, Illinois, June 16, 1966, and was raised in

Miami, Florida. He received his BA in biology from Florida State University in 1991 and

graduated from the University of Florida, College of Veterinary Medicine in 1998. He is

currently employed as the Division of Wildlife Research veterinarian for the Florida Fish

and Wildlife Conservation Commission.