80968 UF MNH cvr.pmd, page 1-2 @ Normalize BULLETIN UNIVERSITY OF FLORIDA GAINESVILLE METHODS OF ASSESSING HEALTH AND DIET OF FLORIDA PANTHERS (Puma concolor) USING MUSEUM SPECIMENS PART I: Osteology as a Means of Assessing Florida Panther Health Laurie Wilkins, Julie M. Allen, Joan Coltrain, Shelly Flanagin, Terry D. Allen, & David L. Reed1 PART II. Stable Isotope Geochemistry: A Method to Evaluate the Diet of Florida Panthers (Puma concolor) Using Museum Specimens Julie M. Allen, Joan Coltrain, Laurie Wilkins, Shelly Flanagin, & David L. Reed Vol. 47, No. 3, pp. 73-108 2007 ™ WILKINS, ALLEN and REED: Florida Panthers 109 The FLORIDA MUSEUM OF NATURAL HISTORY is Florida’s state museum of natural history, dedicated to understanding, preserving, and interpreting biological diversity and cultural heritage. The BULLETIN OF THE FLORIDA MUSEUM OF NATURAL HISTORY is a peer-reviewed publication that publishes the results of original research in zoology, botany, paleontology, and archaeology. Address all inquiries to the Managing Editor of the Bulletin. Numbers of the Bulletin are published at irregular intervals. Specific volumes are not necessarily completed in any one year. The end of a volume will be noted at the foot of the first page of the last issue in that volume. Richard Franz, Managing Editor Erika Simons, Production Bulletin Committee Richard Franz, Chairperson Ann Cordell Sarah Fazenbaker Richard Hulbert William Marquardt Larry Page Tom Webber Irvy R. Quitmyer David Steadman, Ex officio Member ISSN: 0071-6154 Publication Date: November 30, 2007 Send communications concerning purchase or exchange of the publication and manuscript queries to: Managing Editor of the BULLETIN Florida Museum of Natural History University of Florida PO Box 117800 Gainesville, FL 32611-7800 U.S.A. Phone: 352-392-1721 Fax: 352-846-0287 e-mail: drfranz@flmnh.ufl.edu WILKINS, ALLEN and REED: Florida Panthers 73 METHODS OF ASSESSING HEALTH AND DIET OF FLORIDA PANTHERS (Puma concolor) USING MUSEUM SPECIMENS PART I: Osteology as a Means of Assessing Florida Panther Health Laurie Wilkins, Julie M. Allen, Joan Coltrain, Shelly Flanagin, Terry D. Allen, & David L. Reed1 PART II. Stable Isotope Geochemistry: A Method to Evaluate the Diet of Florida Panthers (Puma concolor) Julie M. Allen, Joan Coltrain, Laurie Wilkins, Shelly Flanagin, & David L. Reed TABLE OF CONTENTS Preface...................................................................... 74 Part I. Osteology and Panther Health........................... 75 Abstract......................................................... 75 Introduction.................................................... 75 Methods......................................................... 77 Results and Discussion.................................... 78 Conclusions.................................................... 89 Acknowledgements......................................... 90 Literature Cited............................................... 91 Part II. Stable Isotopes and Panther Diet...................... 99 Abstract......................................................... 99 Introduction.................................................... 99 Methods......................................................... 99 Results and Discussion.................................... 102 Conclusions.................................................... 106 Acknowledgements......................................... 106 Literature Cited .............................................. 106 Key Words: methods, museum specimens, osteology, health, stable isotopes, diet, Florida panther, Puma concolor Wilkins, L, J. M. Allen, J. Coltrain, S. Flanagin, T. D. Allen, and D. L. Reed. 2007. Methods of assessing health and diet of Florida panthers (Puma concolor) using museum specimens. Part I. Osteology as a method of assessing Florida panther health. Bull. Florida Museum of Nat. Hist. 47(3), Pt. I: 73-97 Allen, J.M., J. Coltrain, L. Wilkins, S. Flanagin, and D.L. Reed. 2007. Methods of assessing health and diet of Florida panthers (Puma concolor) using museum specimens. Part II. Stable isotope geochemistry: A method to evaluate the diet of Florida panthers. Bull. Florida Museum of Nat. Hist. 47(3), PT. II: 98-108 Epilogue.........................................................................108 WILKINS, ALLEN and REED: Florida Panthers 75 PART I: OSTEOLOGY AS A MEANS OF ASSESSING FLORIDA PANTHER HEALTH Laurie Wilkins1, Julie M. Allen1,2, Joan Coltrain3, Shelly Flanagin1, Terry D. Allen4, and David L. Reed1 1/ Florida Museum of Natural History, University of Florida, P.O. Box 118700, Gainesville, Florida, 32611 (lauriew@flmnh.ufl.edu, shelly03@ufl.edu, dreed@flmnh.ufl.edu). 2/ Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, Florida 32611 (juliema@ufl.edu) 3/ Department of Anthropology, University of Utah, 270 S. 1400 E., Salt Lake City, Utah 84102 (joan.coltrain@anthro.utah.edu). 4/ Department of Sociology, University of Utah, 270 S. 1400 E., Salt Lake City, Utah 84102 (terry.allen@soc.utah.edu). ABSTRACT Conservation efforts to reverse the negative effects of inbreeding in an isolated population of Florida panthers (Puma concolor coryi) resulted in the release of eight Texas females into Florida in 1995 (Seal 1994; Johnson et al. 1998). Since that time, Florida panthers have shown increased productivity, range expansion, and the reversal of a suite of deleterious morphological and physiological effects of inbreeding. (Land et al. 2005, Pimm et al. 2006). Previously described bone pathologies in the Florida panther may result from a compromised immune system due to inbreeding, poor health related to diet and nutrition, or the presence of previously undetected pathogenic diseases. We examine the current collection of 140 post-cranial skeletons to determine the frequency of trauma, infection, arthritis, and incidence of Harris Lines. Harris lines, visible from X-rays of long bones, represent a cessation of growth due to a major episode of starvation or illness. We compare the population born before and after 1995 to examine changes over a time line that includes genetic, biomedical, and management interventions. Our data support earlier findings that there are idiopathic bone pathologies that exist in the Florida population, and we explore possible causes. Multivariate analysis reveals that Harris Lines and osteopathologies increase with age, and those pathologies affect males more than females, and both show increases after two years of age. There is a reduction in the number and severity of osteopathologies in panthers born after 1995; however, the demographics of our population (as represented in the museum sample) have shifted from an “aged” population to one that includes a disproportionately large number of young animals (<2 years old). It is likely that more than one biological process is operating to produce this result, and the study of osteological material alone cannot provide definitive diagnoses. Advanced studies in the pathology of human arthritis offer intriguing insights, and we expect at least some of their findings to have application in wildlife disease studies. Our results, together with the rich resource of archival material, leads to new cooperative research opportunities between museums, wildlife biologists, and wildlife veterinarians in the efforts to improve the conservation status of Florida panthers. INTRODUCTION Post-mortem skeletal remains reveal a history of activ- ity, injury or traumatic events in the life of an animal. This is expressed by scars, malformations, unusual le- sions, or excessive bone deposition as animals overcome infections, disease, or injury in life. Unusual or abnor- mal osteological features that have been described in panthers include arthritis, evidence of infection, trauma and bone lesions of unknown cause. Harris Lines (HLs), internal osteological features, were also recorded in high frequency in panthers. Duckler and Van Valkenburgh (1998a) showed that 69% of Florida panthers (N=51) exhibited at least one post-cranial osteopatholgy com- pared to 46% in a sample of pumas (N=26) from other locations. In the same study, the prevalence of HLs in Florida panthers was significantly greater (56.9%) than that of non-Florida pumas (27%). They conjectured that the elevated incidence of HLs was due to more regular episodes of poor nutrition, perhaps exacerbated by health problems associated with inbreeding. Harris Lines are visible in radiographs as dense lines of bone deposition in long bones of humans and other mammals (Fig. 1). They record episodes of ar- rested bone growth in young individuals (Park 1964), and have been experimentally induced in rats, rabbits, pigs and dogs by starvation, selective nutrient deficiency, and bacterial inoculation (Harris 1933; Wolbach 1947; Park & Richter 1953; Platt & Stewart 1962; Mays 1995). It was these experimental studies that linked the forma- tion of HLs, with the occurrence of a physical stress (Grolleau-Raoux et al. 1997). In humans, HLs have been associated with episodes of childhood illness (for 76 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) review, see Garn et al. 1968). As a result, they have been used extensively in historical and archeological stud- ies to characterize the health of human populations (Macchiarelli et al 1994; McHenry 1968; Rathbun 1987). Current interpretations differ on the significance of HL formation. Some believe that HLs form as stress lines during accelerated growth spurts rather than as indicators of past illness, trauma, or malnutrition (Alfonso et al. 2005). The alternative explanation—that HLs rep- resent renewed bone growth upon recovery from a se- rious illness—remains well supported (Mays 1995), al- though it has been shown that there is often no one-to- one correspondence between episodes of disease or nutritional deficiency and HL formation (Marshall 1968). Medical monitoring of Florida panthers during the 1980s showed cats to be in variable health declining south- ward towards the Everglades National Park (ENP) and the Fakahatchee Strand State Preserve (FSSP), a con- dition associated with the type and abundance of prey (Roelke 1990), and supported by food habit studies. Scat analysis showed the panthers living north of Alligator Alley were killing predominantly large prey (white-tailed deer and feral hogs), while those panthers living in the FSSP were taking a large number of small prey (rac- coon and armadillo) (Roelke et al. 1986, Maehr et al. 1990). A relationship between diet, health, and pres- ence of HLs may exist in this stressed population, but it is complex and requires further investigation. Studies on the incidence of osteological abnormali- ties in a wild mammal population are rare, and frequently involve the description of a single or few specimens. This is often true because post-cranial skeletons were not archived, and are not available for study. The pan- ther specimens in the FLMNH mammal collection to- day number more than 140 individuals, including those salvaged through mid-2006. This provides an unparal- leled opportunity to expand the earlier study of morphol- ogy conducted by Duckler and Van Valkenburgh (1998a,b) and to further explore the nature of osteopathology in natural populations. The literature on bone pathology in mammals is vast, but often relates to veterinary studies on domestic animals; terminology and the diagnosis of skeletal abnormalities from skeletal as- semblages is difficult and often confusing. Arthritis is defined by the presence of osteophytes, which are bone spicules that develop around joint mar- gins or sites of injury and represent the body’s attempt to repair an injury. Arthritis involves proliferation of bone rather than loss of bone as in osteoporosis. The degree of osteophytosis in humans and animals can vary con- siderably—from single spicules, a ridge of new growth along an area of muscle attachment, to a massive out- growth of bone. The presence of osteophytes is a com- mon indicator of osteoarthritis (OA) or degenerative often age-related joint disease (DJD), which is common in humans (Rogers & Waldron 1995), wild mammals (Fox 1939; Greer et al. 1977) and domestic animals (Jubb et al. 1985). Degenerative joint disease is a progressive condition in which the articular cartilage is slowly de- graded and the surrounding bone reacts by producing osteophytes. The disease affects many animal species, and is common in domestic breeds of cats. However, the presence of osteophytes may be associated with other conditions, as ‘arthritis’ is a general term that in- cludes a broad spectrum of disorders or diseases that has been extensively studied in humans, and is begin- ning to emerge as a field of study in other mammals, as we will discuss in greater detail later. In past studies, joint disorders have been broadly separated into two categories, non-inflammatory and in- flammatory. Osteoarthritis (OA), or DJD, including joint disease resulting from trauma and developmental, meta- bolic, or dietary causes is generally considered to be non-inflammatory (Turnbull & Cowan 1999). Factors Figure 1: X-ray of humerii of Florida panther 15 , UF24563) showing Harris Lines. Harris Lines are transverse lines of bone density that represent a period of arrested growth followed by renewed growth. WILKINS, ALLEN and REED: Florida Panthers 77 cited in pathogenesis of DJD include genetics, abnormal joint alignment, excessive stress, trauma, local inflam- mation, and hormonal influences (McKeag 1992; Lane & Buckwalter 1993; O’Connor & Brandt 1993). Inflam- matory arthritis includes a variety of disorders that typi- cally involve reactive bone formation and fall under the general term spondyloarthropathy (SpA). One particu- lar form of SpA is reactive arthritis (ReA), not to be confused with another type of arthritis, rheumatoid ar- thritis (RA). In humans (ReA) is mediated by a variety of infectious organisms to which panthers may also be susceptible. Variable manifestations of SpA include asymmetrical, pauciarticular (involving less than five joints), peripheral (appendicular) joint erosions and fu- sion, and axial (spine and pelvis) joint inflammations (Resnick & Niwayama 1988; Rothschild & Martin 1993). The use of the term DJD is widespread in the both the wildlife and veterinary medicine literature but the descriptive terminology is confusing as its symptoms over- lap with that of SpA. Non-inflammatory disorders can often lead to secondary inflammations, and inflamma- tory joint disorders commonly result in secondary, often severe degenerative changes (Turnbull & Cowan 1999). According to Rothschild et al. (1998, 2001), it is the more inflammatory form of arthritis, (namely SpA), that is the more likely condition in wild mammal populations, in- cluding large cats, whereas DJD is more often associ- ated with domestic breeds and zoo animals. SpA has been described in hyenas, bears, canids, non-human pri- mates, elephants, large felids, and mammalian and non- mammalian fossils (Rothschild & Rothschild 1994; Rothschild & Woods 1989, 1991, 1992; Rothschild et al. 1993, 1998, 2001). Turnbull and Cowan (1999) described synovial joint diseases in wild cetaceans that included both degenerative and infectious manifestations. Medical management, genetic augmentation, and intervention to increase prey species may have contrib- uted to improved health of Florida panthers over the years, which could reduce the expression of health-related skel- etal anomalies. Our objective was to review earlier stud- ies, investigate a larger sample of cats from a longer time period, and quantify the observed skeletal features to determine if there have been any changes in the fre- quencies found by Duckler and Van Valkenburgh (1998a). Further, using multivariate statistical methodology, we explore the interrelationship of HLs, skeletal anomalies observed per individual, and the degree of severity of those anomalies, and to what extent these vary by age, sex and habitat. Any general deviation from normal bone development is referred to as an Abnormal Osteological Feature (AOF), to distinguish it from the many specific types of pathologies that exist in nature and to have a consistent and easy reference. We hope to learn more about the causes of HLs and AOFs, or at least the con- ditions under which they form, utilizing a large data set and ample life history information about individual Florida panthers. Considerable analogy with human arthritis research was used for insight into panther bone pathology, as oth- ers have done (Rothschild et al. 2001; Turnbull & Cowan 1999), to reveal new directions in arthritis research, and to search for explanations for the high frequencies of HLs and AOPs. The study of osteological material alone cannot provide a definitive diagnosis; therefore this is a first step to categorize an idiopathic disease process that may exist in the panther population of Florida. METHODS HARRIS LINES To document frequency of HLs in Florida panthers, we X-rayed left and right humerii and femora of 69 males and 43 females including animals that died as recently as 2006. HLs were scored when the density line was perpendicular to the long axis of the bone and extended across the entire shaft. This is a more conservative measure as often HLs need only extend one-fourth to one-half way across the shaft of the bone. Ultimately, only humeral HLs were counted, since femora showed very few. Harris Lines do not always occur symmetri- cally, so we totaled the number of HLs present in L and R humerii. Age, sex, year of death, and use area were recorded for each specimen (Appendix 1). Our X-rayed individuals overlapped with those studied by Duckler and Van Valkenburgh, but did not completely duplicate their series. ABNORMAL OSTEOLOGICAL FEATURES Major skeletal elements of Florida panther speci- mens in FLMNH collections including 77 males 50 fe- males, and one unknown (n=128) were inspected and abnormal osteological features (AOFs), including healed fractures, arthritis, infection, unusual lesions, malforma- tions, and unknown pathologies were tabulated (Appen- dix 1). We gained insight into bone pathology by refer- encing studies in archaeology and forensics (Baker and Brothwell 1980; Buikstra & Ubelaker 1994; Rogers & Waldron 1995), paleopathology (Rothschild & Martin 1993), wildlife and veterinary science, and contempo- rary studies in human arthritis, as well as consultation with forensic specialists. Our results are reported and compared to the earlier study by Duckler and Van Valkenburgh (1998a,b). We examined the cranial, axial, pelvic, and appen- dicular skeleton including the feet, and scored any ab- 78 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) normality within each. We recorded the total number of AOFs, being conservative in our estimate. For example, an AOF associated with two or more bones, such as tibia-fibula or humerus-radius-ulna joint was only counted once regardless of the number of affected bones. If there were two lesions on one bone, both would be counted only if they were qualitatively different, or located in a different region of the bone, although we could never be certain of the relationship between any two observa- tions because of possible systemic involvement. The skeleton records a lifetime of insults, and there is no way to know for certain that this scoring system ad- equately distinguished one event from another. No frac- tures that were related to the cause of death were re- corded; that is broken bones without evidence of bone remodeling. An overall severity score (S) was assigned to each specimen, ranging from S1-Mild, S2-Moderate, to S3- Severe, depending on the nature and extent of the anomaly. Measures of severity suggest the greater or lesser expression of a condition or the later phase of a disease. While neither of these may have any basis in clinical practice (Rogers & Waldron 1995), they do pro- vide a basis for comparison. A severity score of S3 (the most severe case) was assigned only in cases where bones were broken or the deformation from normal state was extreme, and potentially crippling, the latter having been noted either in life through observations and re- ports, or during necropsy. Since many skeletons had more than one incident that ranged in severity from S1 to S3, the highest single score was selected, since that one would have the greatest potential threat to the sur- vival of that animal. PANTHER STUDY GROUPS In many cases, the ages calculated for Florida pan- thers were estimates, so age classes were established based on several references, including degree of fusion of cranial sutures and epiphyseal closure, direct com- parison with animals of known age, and estimates pro- vided in FWC annual reports (Land et al. 2005). Ani- mals were grouped into five age classes (Class I = <1 year, Class II = 1-2 years, Class III = 2-4 years, Class IV = 4-10 years, and Class V = > 10 years. To gain an understanding of the relationship of health parameters (HLs, AOFs) to regions of Florida (north or south), we assigned a “Use Area”(UA) to each animal based on published reports or, in the case of panthers not radio- collared by the FWC, the location where they were found dead. Use areas were defined UA1) north of Interstate 75 (former SR 84 or Alligator Alley), and UA 2) south of Interstate 75 including southern Big Cypress National Preserve (sBCNP), Fakahatchee Strand State Preserve (FSSP), and the Everglades National Park (ENP)(see map in Part II). Past health and diet studies of panthers frequently delineated three distinct regions by consider- ing ENP a separate region, but sufficient samples did not exist, so ENP cats were grouped into UA2. ANALYSES The dependent variables in the study are total num- ber of abnormal osteological features (AOFs), severity index (SI) of AOFs, and HLs. The independent vari- ables were sex, age class and use area. The model used to test the dependent variables included all three independent variables and all possible interactions. A Multivariate Analysis of Variance (MANOVA) was performed simultaneously testing all three dependent variables against the independent variables using SAS (SAS Institute Inc., Cary, NC). Once significance was established with the MANOVA, individual ANOVAs were run on each independent and dependent variable. A student’s t was used to find differences between Age Class groups whenever significance was established for that variable. All of the individual ANOVAs were ana- lyzed using JMP version 5.0 Statistical software for the Macintosh (SAS Institute Inc., Cary NC). The relationships between the three dependent variables (HLs, AOFs, and SI) were analyzed using re- gression analysis. Animals with no osteopathologies were removed because these animals also had a scor- ing of zero for the AOF severity index. Therefore, only animals with at least one AOF were used to examine trends between the dependent variables. To determine the effect on HL or AOF frequency resulting from the introduction of Texas cats in 1995, we compared the prevalence of HLs and AOFs of animals that were born prior to 1995 (HL n=63, AOF n = 79), to those born since that time (HL n=49, AOF n = 49) using ANOVA. RESULTS AND DISCUSSION The MANOVA testing the differences among total AOFs, Severity of AOFs, and total HLs on sex, age class, and use area shows a significant multivariate ef- fect for sex and age class. This test fails to reveal any significant multivariate effect for use area or any inter- actions of the three independent variables (Table 1). HARRIS LINES The average number of HLs increases with each Age Class (AC) for all animals combined (Fig. 2). There is a continuous increase in number of HLs with each successive AC and overall significant differences were found between the ACs (ANOVA; F4,107 = 4.82, p = WILKINS, ALLEN and REED: Florida Panthers 79 0.0013). A significant increase exists from AC-I to AC- IV as well as from AC-IV to AC-V. There is no differ- ence between males and females with respect to HLs, nor was there any significant difference between the average number of HLs for animals from the north, above SR 84, and those from the south, including sBCNP, FSSP and the ENP (MANOVA results; Table 1). The apparent continual accumulation of HLs throughout life is an unexpected result. Harris Lines develop at the metaphases during growth before the epiphyseal plate fuses to the long bone, at which point bone growth stops. For panthers, this would be approxi- mately between the ages of three and four years. Be- cause bone is remodeled over time, it is also expected that evidence of HLs would disappear with age as bone continuously remodels, so an older animal should have fewer HLs rather than more. One possibility for the continued visibility of HLs in older animals is that previ- ously formed HLs become easier to detect as cats age, perhaps due to a reduction in the thickness of the bone cortex. This has not been studied, however, it is pos- sible to measure cortical bone thickness in individuals that vary in age to determine if this is the case. (A. Falsetti, C. A. Pound Human Identification Lab, pers. comm.). If it is assumed that HLs are indeed an indica- tion of stress in the population, then a likely explanation is that the older animals in our study experienced greater health-related stress at a younger age than those in the younger age classes, a possibility also noted by Duckler and Van Valkenburgh (1998a). This might suggest that health or living conditions have improved since the mid- 1980s. This is consistent with information from diet and prey studies conducted in the mid-1980s in the sBCPR and FSSP that reported that animals in the south were eating fewer deer and more smaller prey, and that the general health of animals living in the Fakahatchee was poor (Roelke et al. 1986, Maehr et al. 1990, Roelke 1990). This time period during the 1980s also reflects a time when deer hunting in the FSSP was legal, but later banned, and therefore panthers and hunters may have been competing for the same large prey species with the result that panthers in FSSP were eating small prey rather than deer and hog (Roelke 1990). This too is a complex issue because many factors affect prey den- sity. Prior to 1980s there was no intentional manage- ment on public lands to increase prey (such as deer) for panthers. During the 1980s, actions were taken to re- duce access to public lands, reduce hunting pressure and harvest on white-tailed deer and hogs, reduce the use of hunting dogs, and protect does and fawns (Schortemeyer et al. 1991, Beier et al. 2003). Addi- tional factors affecting deer densities are length and in- tensity of hydroperiod in the ENP that might affect fawn survival and variable quality of vegetation and soil types (Land 1991, Fleming 1994). Improvements in prey base throughout the range of panthers would contribute to a healthier population. Contemporary arguments exist that HLs do not form as a result of pathological stress or disease, but rather may reflect accelerated growth during specific Table 1: MANOVA results for Florida panthers from the Florida Museum of Natural History. The MANOVA examined three dependent variables; total AOFs, severity of AOFs, and Harris Lines. Variables Wilks’ Lambda F statistic df p Sex 0.90 3.14 3/88 0.0292 Age Class 0.59 4.29 12/233.12 <0.0001 Age Class * Sex 0.83 1.37 12/233.12 0.1789 Use Area 0.98 0.46 3/88 0.7006 Use Area * Sex 0.98 0.50 3/88 0.6859 Age Class * Use Area 0.92 0.61 12/233.12 0.8299 Age Class * Use Area * Sex 0.96 0.28 12/233.12 0.9923 Figure 2: Mean number of Harris Lines for Florida panthers at each age class. Letters represent groups that are significantly different. Sample sizes are; Age class I (n=13), II (n=23), III (n=27), IV (n=31), V (n=18). 80 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) early periods of development (Alfonso et al. 2005). Many of the HLs observed were faint, spatially clustered and appeared more frequently in proximal humerii close to the epiphyseal closure. The clustered nature of the HLs may reflect many individual stresses associated with rapid growth during a brief period of time. It is unlikely that this study will clarify the debate on the cause of HLs in vertebrates at least not without further research. However, it should be noted that serious health incidents nearly always result in transverse lines of considerable density, and such lines tend to remain detectable despite subsequent bone remodeling or advanced age (Park 1964, Maat 1984). Dense lines in panthers frequently occur in the humerus anywhere from slightly distal to the midpoint up to the upper proximal portion of the limb bone. No dense lines in the lower quadrants were observed. If HLs are caused by pathological stress or nutritional de- ficiencies, then those events may not occur during the earliest months after birth, but rather after cubs become independent and begin to disperse at approximately 14 months of age. This activity might be more stressful, especially for males who move a greater distance than females, who must compete with resident males in other locations (Maehr et al. 2002). Harris Lines formed in very young animals are more likely to disappear as the bone is continuously remodeled during the earliest months of growth. As pointed out by Duckler and Van Valkenburgh (1998a), HLs are more likely to be retained in the adult skeleton when they form close to the time of epiphyseal closure and growth termination. Duckler and Van Valkenburgh (1998b) also showed that pumas throughout their range have higher incidence of HLs compared to other extant species, including mule deer (Odocoileus hemionus), bobcat (Lynx rufus), and gray wolf (Canis lupus). This may be due to the stress to the bone brought about by one or a combination of larger size (compared to the wolves), and the reckless nature of their hunting style, in which leaping over boul- ders, jumping from trees, and incredible spurts of speed are legendary (Sunquist & Sunquist 2002). However, why Florida panthers have more HLs than puma from other geographic locations remains uncertain. A long history of poor health as a result of diet, as well as del- eterious consequences of inbreeding manifested as car- diac defects, and high pathogen/parasite loads increased the potential for illness or disease and HL formation (Duckler and Van Valkenburgh 1998b). This remains a plausible explanation. A superficial review of the life history of several young cats does not support the conventional view that HLs form as a result of one particular stressor or star- vation. For example, Florida panther 22 was captured by FWC at six months old, subsequently abandoned by her mother, and was later recaptured by FWC in a starved and dehydrated condition. She was rehabilitated, re- leased, and again in 3-4 months, as a result of another FWC capture attempt, she was separated from her mother and was recaptured again in a debilitated and dehydrated condition. She was brought into captivity and not released until 2 years later (M. Roelke, pers. comm.). In spite of two traumatic separations from her mother, two bouts of starvation, two capture events, and a two-year period in captivity, there is no evidence of HLs in this individual. Her sibling, FP 23 also captured twice had distinct HLs. Florida panther 8, who later in life was captured and found to be underweight and in poor health, had HLs, but they were in the middle por- tion of the limbs, indicating that the cause of the HL occurred when she was very young. These examples demonstrate that it is not possible to make a direct cor- relation between episodes of starvation and the pres- ence of Harris lines, particularly in older individuals. HLs reportedly form as a result of a variety of infectious diseases, as well as protein or vitamin defi- ciencies. There is also evidence to suggest that they may form in humans as a result of exposure to mercury and other heavy metals (Raber 1999). A study examin- ing the presence of HLs as they relate to mercury levels recorded in individual panthers is underway (M. Cunningham, pers. comm.). The importance of HLs as indicators of pathologi- cal stress has gone in and out of vogue over the past 50 years. Bone formation and homeostasis is a dynamic process, and many physiological processes affect bone health. Earlier research (Duckler and Van Valkenburgh 1998a) has shown that prior to 1995, Florida panthers had significantly more HLs (56%) than non-Florida popu- lations (27%) (Table 3). We are unable to compare our results directly to theirs since we used total HLs for each animal specimen rather than presence per side. In addition, we did not have the opportunity to examine X-rays of the non-Florida sample. In our results, an ANOVA testing for HL differences between animals born before and after 1995 showed no significant differ- ence (F1,110 = 1.31 p = 0.2554) although there is a slight trend showing a reduction in the average number of HLs of post-1995 animals, (1.53) compared to the pre-1995 animals (1.92). Presence of HLs in Florida panthers has not diminished over time in any significant way and HLs continue to be abundant in all age classes. Future research will investigate the relationship between HLs and variables such as overall health, mercury levels, and specific biomedical conditions reported for panthers. WILKINS, ALLEN and REED: Florida Panthers 81 ABNORMAL OSTEOLOGICAL FEATURES The total number of AOFs was significant for both sex and age class, in the MANOVA analyses (Table 1). Males had more AOFs than females (ANOVA; F1,126 = 4.45, p = 0.0369; Fig. 3), and older animals had more AOFs than younger animals with the total number in- creasing with each successive age class (ANOVA; F4,123 = 12.84; p < 0.0001; Fig. 4) There were no significant results in the interaction between any two variables and our dependent variables in the MANOVA. However, when ANOVAs were run for just severity of AOFs, use area, and age class (Fig 5), a significant interaction was found (F9,116 = 2.55 p = 0.043; Fig. 5). These results suggest that a trend exists, but the original MANOVA is not sensitive enough to detect this. Alternatively, it could represent a type I error, but because results are significant it is necessary to mention the possible trend. The severity of the AOFs in Use Area (UA) 2 are more variable than UA 1, but generally animals in this group also have more severe AOFs. Severity (and occurrence) increases between AC-II and AC-III, between ages of approximately two to four. A nearly significant interaction was also found between sex and age class with severity index (SI) of AOF’s (F9,115 = 2.36 p = 0.057; Fig. 6). Males and females have a similar low SI in AC-I and AC-II, but males increase dramatically in the severity of AOF’s compared to females in AC-III and AC-IV. This corre- sponds to the period of time that males are dispersing and attempting to establish a territory and home range. Figure 3: Mean Abnormal Osteological Features (AOFs) for female (n = 50) and male (n = 78) Florida panthers with error bars. Males have significantly more AOFs than females (ANOVA; F1,126 = 4.45, p = 0.0369). Figure 4: Mean AOFs for Florida panthers for each age class with error bars. Letters represent significant differences between groups. Sample sizes are: Age Class I (n=14), II (n=27), III (n=34), IV (n=35), V (n=18). Figure 5: Interaction plot between Florida panthers from the two different use areas. Each point is a mean for each age class. Panthers in age class three show a significant difference between the two use areas (ANOVA F9,116 = 2.55 p = 0.043) Figure 6: Interaction plot for female and male Florida panthers in each age class. Females and males from the 3rd, 4th and 5th age classes are significantly different from each other (ANOVA F 9,115 = 2.37 p = 0.057). 82 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) but when we repeat the analysis after removing AC-V, our results remain significant for total AOFs (ANOVA; F1,108 = 9.61 p = 0.0025). This leads us to conclude that there is a trend towards a reduction of AOFs and their severity since 1995. Although no correlation exists between Harris Lines and total AOFs (Fig. 8A,B), a correlation exists between the number of AOFs and the severity of AOFs (Fig. 8C). This correlation is expected given that an increase in the number of AOFs will increase the prob- ability that at least one of them is severe. Panthers over the age of two born in Florida over the last 50 years, show a range of mild-to-severe, and potentially debilitating, osteological conditions (Appen- dix 1). These include trauma (breaks and bite wounds), arthritis, periostitis (disease of the periostium), enthesitis (inflammation of osseous attachment of tendons, liga- ments, synovium) and other unidentifiable infections fre- quently observed in the feet, forearms, and axial skel- eton. The overall incidence of fractures and other trauma remain high when compared to the population of non- Florida panthers analyzed by Duckler and Van Valkenburgh 1998. (Table 3). The overall prevalence of all pathologies in our sample was 60.8% in the pre-1995 sample compared to 69% reported by Duckler and Van Valkenburgh (1998a). However, our scoring protocol differed in that we counted number of AOFs rather than a type and presence/absence per side. This resulted in more incidents of AOFs in our study. We also did not consider HLs in our final totals of AOFs, thereby poten- tially lowering the score. We preferred to treat HLs separately since there is disagreement as to whether HLs are only associated with illness or injury. Further, we hoped to learn more about the possible relationship of one to the other through these analyses. Regardless of how pathologies were counted, both studies found a greater than 50% incidence of abnormal osteological features in the population of Florida panthers. This is far greater than any published study of abnormal bone development in any wildlife species, although those stud- ies are themselves very rare. Pathologies were less frequent in the post-1995 sample (n=49) with 18 indi- viduals (36.7%) exhibiting one or more pathology com- pared to 48 (60.8% pre-1995). The fraction of the post- 1995 population with pathologies is significantly lower than the pre-1995 sample. However, the proportions of old versus young animals in the two samples vary con- siderably. There is a 34% increase in animals under two years of age in the post- 1995 sample, and it has already been demonstrated that injuries and other or AOFs accumulate with age (Table 3). Maehr et al. (2002) describe the process as beginning at approximately 14 months of age and continuing for 7.0 months for females and 9.6 months for males with males’ efforts frequently frustrated by insufficient vacant range or range containing no individuals of the opposite sex. Independence and dispersal of young cats can, and prob- ably does, increase food stress as well as encourage, especially in males, male-male conflicts. As males dis- perse, they also have a greater chance of injury (and death), thus accounting for both the increase in young animals in our collection, and increased evidence of trauma and AOFs. Our sample consists of animals born prior to 1995 (n=79) and those born since 1995 (n=49; Table 2). There has been a significant reduction of both the number (ANOVA; F1,12 = 13.53, p = 0.0003; Fig. 7) and severity (ANOVA; F1,124 = 6.97, p = 0.0093; data not shown) of AOFs since 1995 in a sample that includes both males and females. There is only one animal in AC- V that was born after 1995 (n = 1; Table 2). This could bias these results because AOFs also increase with age class, Figure 7: Mean AOFs and error bars for Florida panthers born before 1995 (n = 79) and after 1995 (n = 49). Panthers born after 1995 have significantly fewer AOFs (ANOVA; F1,126 = 13.53, p = 0.0003). Table 2: Number of Florida panthers in age class for animals born before and after 1995. Age Class Pre-1995 Post-1995 Total 1 6 8 14 2 9 18 27 3 21 13 34 4 26 9 35 5 17 1 18 WILKINS, ALLEN and REED: Florida Panthers 83 The most abundant osteological expression was that of arthritis, which was present in 53.2% (n=42) of the sample of animals born before 1995, but only 30.6% (n=15) of those born after 1995. It might have been easy to dismiss the severe arthritis we observed as age- related, since many of the specimens acquired in the 1980s and early 1990s represent an “aged population” (Ballou et al. 1989; Roelke 1990; Duckler & Van Valkenburgh 1998a). However, arthritis, as the accu- mulation of osteophytes, even in an incipient form, was present in cats of all ages, except the very youngest (Age Class I). Young animals (those younger than two years of age) did not show significant arthritic lesions, and there are considerably more young animals (53.1%) in our post-1995 sample. Age is a significant factor to be considered in the interpretation of these results, as in- sults to the skeleton continue to accumulate through life. Some types of lesions were more prevalent, whereas others were more notable because of their se- verity. Among the most common AOFs were inflam- mation and osteophytosis, an inflammatory arthritis, in forelimbs, including elbow and wrist. A 3-year old male (FP 89), reportedly small for his age, had extensive pro- liferation of bone around the articular surfaces of both the left and right humerus/radius/ulna joints, which was also observed in life as an open wound at the elbow (M. Cunningham, pers. comm.) (Fig. 9). A similar, but even more extreme case is that of FP16 from Dade County. Evidence of osteoarthritis was present in the spine and feet of this 14-year old male, however, all bones of the forelimb showed an advanced joint disease, possibly caused by a broken right radius/ulna, which calcified into a large mass in healing (Fig. 10A). Deep eburnations visible in the trochleae of both humerii and the trochlear notch of both ulnae reflect the loss of the protective cartilage resulting in bone-on-bone contact (Fig. 10B). Long-term instability and/or inflammation were indicated by the excessive bone deposition around both joints sug- gesting a debilitating and presumably painful condition. In addition, the distal fibulae where they attach to tibiae, both appeared to have been deformed or possibly bro- ken, a condition that is difficult to explain (Fig. 10C). While these were the most extreme cases, there were other cats with less severe inflammations, but that also involve the olecrenon process, the attachment site of the complex triceps brachii muscle. Often the condi- tion was expressed in both limbs, which might suggest a chronic front limb dysplasia creating the potential for instability, injury and inflammation. Some of these same animals also showed osteophyte formation at distal ra- dius/ulna above the level of the wrist, which may be a related condition. These cases, particularly in the young panther FP89, where there was no obvious evidence of a wound or injury that could result in an infection, alerted us to the possibility that there was a predisposition to inflammation at certain sites. This led us to scrutinize those sites (e.g., elbow joints) where we observed the early signs of inflammation or distortion. One Florida panther (FP 2; UF 20777), shows areas of inflammation at the proximal ulnae, and, both distal left and right ulnae show evidence of injury with subsequent osteophyte for- mation (Fig. 11). FP 205 (UF26520), approximately 1-1/ Figure 8: Correlations between total number of Harris Lines (A), total AOFs (B), and severity of AOFs (C) for Florida panthers. Points were jittered (offset) by adding a random number between –1 and 1 to each point so that the points don’t lay on top of each other. Animals without any AOFs were removed from the analysis because they also had a zero Severity Index, which would influence the regression line. 84 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) Ta bl e 3. N um be r a nd p er c en t o f i nd iv id ua ls w ith A O Fs a nd H ar ris L in es in 2 p op ul at io ns o f P um a co nc ol or o ve r t im e an d a co m pa ris on o f t w o st ud ie s o f Fl or id a pa nt he rs . To ta l i nd iv id ua ls a ( w /c om bi ne d3 O st eo pa th ol og ie s/ A O Ps ) Sa m pl e (N ) A rth rit is In fe ct io n Tr au m a U nk no w n To ta l i nd iv id ua lsa H ar ris L in es 4 S3 se ve rit y5 # ag ed 2 y rs Pr ev io us S tu dy 1 Fl or id a Sk ul l 53 0 1 (2 % ) 1 (2 % ) 2 (4 % ) 4 (8 % ) 0 Sk el et on 51 9 (1 8% ) 4 (8 % ) 4 (8 % ) 6 (1 2% ) 35 (6 9% ) 29 (5 7% ) N on -F lo ri da Sk ul l 44 3 0 3 (0 .7 % ) 3 (0 .7 % ) 8 (2 % ) 14 (3 % ) 0 Sk el et on 26 3 (1 2% ) 0 4 (1 5% ) 5 (1 9% ) 12 (4 6% ) 7 (2 7% ) Th is S tu dy 2 Fl or id a Sk ul l + Sk el et on AO P/ H L Pr e- 19 95 79 /6 3 42 (5 3. 2% ) 9 (1 1. 4% ) 12 (1 5. 2% ) 12 (1 5. 2% ) 48 (6 0. 8% ) 42 (6 6. 7% ) 13 (3 1. 0% ) 15 (2 3. 8% ) Po st -1 99 5 49 /4 9 15 (3 0. 6% ) 1 (2 .0 4% ) 5 (1 0. 2% ) 3 (6 .1 % ) 18 (3 6. 7% ) 29 (5 9. 2% ) 5 (1 7. 2% ) 26 (5 3. 1% ) To ta l 12 8/ 11 2 66 (5 1. 6% ) 91 (8 1. 3% ) 1 D uc kl er a nd V an V al ke nb ur gh 1 99 8a 2 T he tw o st ud ie s u se d di ff er en t p ar am et er s i n m ea su rin g pa th ol og y an d ar e no t d ire ct ly c om pa ra bl e; o ur d ef in iti on fo r a rth rit is w as m or e in cl us iv e of m in or sk el et al c ha ng es 3- 4 H ar ris L in e co un t i n cu rr en t s tu dy is n ot in cl ud ed in O st eo pa th ol og y (a s i t i s i n th e D uc kl er & V an V al ke nb ur gh st ud y) . W e do n ot c on si de r H Ls to b e str ic tly a fu nc tio n of p at ho lo gy , s ee te xt . 5 Th e hi gh es t m ea su re o f s ev er ity re pr es en ts b ro ke n bo ne s, ad va nc ed d eb ili ta tin g ar th rit is , o r o th er e xt re m e de vi at io ns fr om n or m al b on e de ve lo pm en t; th e pe rc en ta ge s ho w n is b as ed o n th e nu m be r o f i nd iv id ua ls w ith a se ve re c on di tio n am on g th e to ta l n um be r o f n um be r o f i nd iv id ua l a ni m al s e xp re ss in g on e or m or e pa th ol og ie s. WILKINS, ALLEN and REED: Florida Panthers 85 2 years old, shows osteophyte formations at the metaphases (Fig. 12). Note that FP205 was not included in the statistical analyses, because he spent time in cap- tivity. However, it is noteworthy that this animal died of an infection due to esophageal laceration, and the osteo- phyte formation visible in the photograph may be a sys- temic reaction to that infection. A second very distinctive osteopathology worth noting was a severely degraded, porous, and rugose dis- tal ulna in panther FP 10 (UF23986), who was killed by another Florida panther. The remnants of a periostitis infection, suggested by a layer of black film that cov- ered most of the cleaned limb bones (Fig. 13) might have been a contributing factor. There were several young animals that exhibited a very porous state in the distal radius/ulna region of the forearm. Without knowing more about the developmental process we cannot say this is abnormal, only that young cats were vulnerable to os- teophyte formation and infections in the distal ulna. Frequencies of fractures and other trauma are high compared to the population of non-Florida panthers and much higher than in other vertebrates such as deer and wolf (Duckler & Van Valkenburgh 1998a,b). Few com- parative studies exist of traumatic, degenerative or de- velopmental lesions among wild carnivores or any mam- mal species. Among the few is a study of wolves (Ca- nis lupus) and coyote (Canis latrans) in Saskatchewan (Wobeser 1992). Wolves showed a much greater num- ber of broken bones (22.8%), but degenerative joint dis- ease, involving the spinal column and limb joints similar to that reported here, was found in only seven wolves and two coyotes. As with panther FP16 (UF29821), the articular cartilage of a single wolf was eroded from the condyles of both femurs with eburnation of the underly- ing bone and extensive periarticular osteophytes were present. One would expect a greater number of injuries in carnivores, such as wolves and puma, than other mam- mals because hunting is a dangerous activity. Although the osteopathologies of non-Florida wild living puma were high (n=12, 46%), they were not as high as those ob- served in the Florida population (Duckler and Van Valkenburgh 1998a). Now, ten years later with a sample Figure 9: Example of severe inflammatory arthritis in ca. 3-year old Florida panther (FP89, UF30064) that died of intraspecific aggression. Left are the right and left humerii, and corresponding ulnae are seen at the right. In life an open wound was observed at the elbow. 86 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) twice that of Duckler and Van Valkenburgh’s study (1998a), we do see a decline in trauma and arthritis. However, once again, it is necessary to consider the proportionate difference in age classes. Following Duckler and Van Valkenburgh (1998a,b) we have docu- mented a high incidence of injury, arthritis and other pa- thology associated with the skeletons of Florida pan- thers. We are not able to attribute them to any specific type of arthritis, since so many have been described (see following discussion of human arthritis studies) but it is apparent that some level of infection is operating, and it is likely that more than one causal agent exists. In sev- eral specimens, the degree of osteophytosis alone, whether initially due to an injury with secondary infec- tion, would suggest there is a tendency for a systemic and inflammatory condition to prevail. The consistent involvement of the enthesis of the olecrenon process and the distal radius/ulna suggests abnormal joint align- ment or ligamentous instability, particularly because these were often symmetric. This may also cause inflamation. Another consideration is that this front limb joint is more vulnerable to injury since it bears significant stress in many locomotor activities (see McGonagle et al. 2001 below). Thirty-eight panthers have been killed by in- traspecific aggression (Land et al. 2005); several of these were severely debilitated. NEW ARTHRITIS RESEARCH Before we attempt a discussion of possible causes AOFS, we would like to present a summary of the in- tensive research in human arthritis during the last two Figure 10: All bones of forelimb show advanced joint disease in this 14-year old male (FP16, UF29821), probably initiated by the broken right radius/ulna (A). Deep eburnations visible in the trochleae of both humerii and the trochlear notch reflect the loss of the protective cartilage, resulting in bone-on-bone contact (B). Distal fibulae, at the attachment site of tibiae, both appeared to have been deformed or previously broken (C). WILKINS, ALLEN and REED: Florida Panthers 87 decades. This provides new information and intriguing insights into the pathogenesis the spondyloarthropathies (SpA), also referred to as arthropathies. References to SpA in wildlife studies are rare, but do exist (Turnbull and Cowan 1999), and recall that they have been identi- fied in museum specimens (Rothschild and his colleagues, op cit.). Spondyloarthpathies include a diverse suite of related conditions in humans, including ankylosing spondylitis, reactive arthritis, psoriatic arthritis, and in- flammatory, bowel-disease (Benjamin & McGonagle 2001). Collectively these arthropathies are character- ized by inflammatory arthritis, extra-articular inflamma- tion, preceding bacterial infection, seronegativity for rheu- matoid factor, and a strong genetic association (HLA- B27) (Dougados et al. 1991; Calin & Taurog 1998). In humans, an important factor associated with the sus- ceptibility of an individual to reactive arthritis is HLA- B27. It appears that B-27 positive individuals are af- fected more severely, although the pathogenesis is still not fully understood (Toivanen and Toivanen 2004). Today SpA is commonly referred to as enthesopathy because of the involvement of the enthesis, the insertion site of a tendon, ligament, or articular capsule into bone. Enthesitis, the inflammation of an enthesis, is believed to be a unifying concept for SpA (McGonagle et al. 1998). Numerous enthesitic arthritic lesions, some incipient and others grotesquely abundant, were observed at the olecrenon process and distal radius ulna of several young panthers. Enthesitis can accompany many disorders, including traumatic, degenerative, inflammatory, endo- crine, and metabolic conditions. In some cases, enthesitis represents the initial or predominant manifestation of dis- ease (Resnick & Niwayama 1983). Furthermore, one aspect of SpA, namely ReA, (reactive arthritis) is now considered in humans to be a disease, triggered by a host of infectious agents including bacteria, parasites, and viruses (Toivanen & Toivanen 2001a,b). The in- flammatory expression is the interaction between the infectious agent and the host immune response (Schoen 2000). A hypothesis in the study of psoriatic arthritis is that enthesitis arises at sites of high shear and compres- sion forces, with the additive interaction between me- chanical stress, microtrauma, tissue repair mechanisms, and bacterial molecules variably leading to inflammation (McGonagle et al. 2001). Whether it is possible to extend human research to panthers is as yet unknown. The tendency for front limb inflamation at the enthesis of the humero-radius ulna joint may be due to the biomechanical stresses of the front limbs associated with puma hunting behavior and occasional injury. However, it is also well known that panthers are immuno-compromised and vulnerable to a variety of pathogens (bacterial, viral, and parasitic) possibly as a result of low genetic diversity (Roelke et al. 1993a,b; Glass et al. 1994; Rotstein et al. 2000). Pan- thers exhibit a variety of congenital abnormalities, such as atrial septic defect and cryptorchidism related to low genetic variability (Beier et al. 2003; Pimm et al. 2006). Elbow dysplasia in dogs is influenced by multifactorial processes including genetic predisposition, and if un- treated is known to progress to crippling osteoarthrosis (Pool 2002). Dysplasia and/or the increased suscepti- bility to osteological inflammation as a result of injury or infection could be another previously unsuspected mani- festation of panther inbreeding. Further, inflammations associated with SpA, such as those identified in the el- Figure 11: Areas of unusual rugosity or inflamation at the proximal ulnae and osteophyte formation at the distal, possibly due to injury or instability caused by dysplasia (FP 2, UF20777). 88 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) bow joint of panthers in this population, have been de- scribed in other wild mammals, including cetaceans, and are considered to be debilitating and a significant mor- tality factor if allowed to progress (Turnbull and Cowan 1999). Veterinary involvement with the panther began in 1983. Since that time, biomedical research to further the understanding of disease, nutrition, and reproductive physiology has been an integral part of the Florida pan- ther recovery efforts (Roelke 1990, Dunbar 1994). Among the many thousands of biological samples taken over the years including blood, urine, skin biopsy, feces, hair, saliva, viral and bacterial culture swabs, one is of particular interest here; namely Feline Syncytia-forming Virus (Roelke, pers. comm.). Feline Syncytia-forming virus (FeSFV) is common in healthy as well as sick do- mestic cats, but has been reported in conjunction with chronic progressive polyarthritis. Pedersen et al. (1980) reported that in a study 20 domestic cats with CPA be- tween 1.5 and 5.0 years of age, only males were af- fected, and two distinct forms of the disease were mani- fested. The following is their actual account: “The most prevalent form was characterized by osteopenia and periosteal new bone formation surrounding the affected joints. The second form was characterized by severe subchondral marginal erosions, joint instability, and deformities. The periosteal proliferative forms resembled Reiter’s arthritis of man, and the deforming type resembled human rheumatoid arthritis. The disease began as tenosynovitis and synovitis, with subsequent changes in the articular cartilage and periosteal bone. Histopathologic changes in these cats were similar to those occurring in both chronic Reiter’s and rheumatoid arthritis of man. Chronic progressive polyarthritis of cats was not caused by identifiable bacteria or Figure 12: 1.5 year old Florida panther (FP 205, UF26520), shows early osteophyte formation at the metaphysis. This animal died from an infection due to esophageal laceration. Figure 13: Distal ulna is abnormally porous and rugose; possible cause is a perostitis, an infection of the periostium, suggested by a layer of black film that covered most of the cleaned limb bones (FP 10, UF23986). WILKINS, ALLEN and REED: Florida Panthers 89 mycoplama, but was etiologically linked to feline leukemia virus (FeLV) and feline syncytia- forming virus (FeSFV) infections.” Pedersen et al. (1980) postulated that polyarthritis was an uncommon manifestation of FeSFV that occurred in predisposed male cats. Feline leukemia virus may not have been directly involved in the disease, but may have acted in some way to potentiate the pathogenic effects of FeSFV. Of all panthers tested for FeSFV from 1987- 1992, 59% were found to be infected with FeSFV (origi- nal number not disclosed; Dunbar 1994). Sixteen of those panthers reside in the FLMNH collection and some of the males showed signs of severe arthritis, whereas females were less affected. Seven males (FP7, 10, 12, 13, 16, 17, and 20), demonstrated the following arthritis or unknown infections: SI of 3 (n=2), SI of 2 (N=4), and SI of 1(N=1), and five of seven males had three or more episodes of arthritis and infection that we would describe as arthritic, infectious, or having a component that would coincide with a description of enthesititis (inflamation in region of enthesis). Of the eight females represented in our collection, most showed no evidence (N=2), or mild arthritis (N=3), moderate infection on the rear feet (N=1), and more extreme episodes were apparent in old fe- males (N=2, FP18 and 21, 11 and 14 years of age, re- spectively). It is difficult to come to any conclusions based on this small sample, but males had both more AOFs and more severe AOFs. Tests for FeSFV were discontinued after 1994, however, archived tissue samples exist and it may be worthwhile to explore this medium as a possible contributing factor to the high incidences of AOFs in Florida panthers. Environmental contaminants such as methylmer- cury might further compromise the animals’ ability to resist disease (Roelke et al. 1991). As well, they may be a contributing factor in the formation of Harris Lines and/or other osteological pathologies. Elevated levels of mercury have been reported in Florida panthers (Newman et al. 2005), and the death of at least one animal (FP27, UF 24557) from the Everglades was sus- pected to be the result of mercury toxicosis. Mercury is known to interfere with bone metabolism and calcium homeostasis (Suzuki et al. 2004), although it is not known if it exacerbates the inflammatory process. Presence of mercury in south Florida environments may also eventu- ally explain the high incidence of Harris Lines. Dense metaphyseal banding has many possible causal agents, but heavy metals including lead and mercury, can induce physiological changes that result in increased calcium deposition (Raber 1999). An examination of whether Mercury plays a role in the high number of osteopathologies in Florida panthers is warranted. CONCLUSIONS AND RECOMMENDATIONS We present preliminary results of an exploration into presence and causes of abnormal osteological fea- tures, some of which may have a pathogenic basis, with respect to a timeline that includes genetic out-breeding, biomedical monitoring, and habitat and prey-base im- provements. Our study demonstrates that HLs accu- mulate over time throughout all age classes, but there is no difference in the frequency of HLs between males and females, no difference in the number of HLs ob- served on cats living in the north versus the south, nor is there any significant difference between pre-1995 and post-1995 populations with respect to number of HLs. Harris Lines remain common in Florida panthers, how- ever their cause is enigmatic and somewhat controver- sial. Food stress, illness, and infection cannot be elimi- nated as possible causes for HLs. Exposure to heavy metals, including Mercury, are known to cause HLs in humans as well. We cannot eliminate the possibility that at least some of the HLs are a response to acceler- ated growth at certain times in the growth cycle. Inas- much as HLs were also frequent in Puma from other geographic regions, and in much higher frequencies than in other large mammals in general. Another plausible explanation for HLs might be high compression forces operating on the forelimbs. As we are able to gather further information about causes of AOFs, we may be better able to understand HLs and their relationship to bone pathology. Osteopathology, and specifically joint disease, ex- pressed through the formation of arthritic lesions in the forelimbs, of Florida panthers appears to exist out side the normal limits of a healthy population. Both AOF numbers and severity increase with age, with males showing a more dramatic increase than females, which may correspond to the risks that dispersal places on young animals, particularly males. The potential causes of AOFs are multiple, complex, and probably interre- lated. Multiple physiological inflammatory responses to a broken bone are likely, as it presents an opportunity for a pathogenic invasion (Woodard & Riser 1991). We have seen that a break can create a systemic response, with equally well developed and even grotesque arthritic lesions on the opposite limb. Of course, a broken bone creates instability, not only in the damaged area but on other joints as well. The forelimb has shown a much higher incidence of osteophytic development than other limbs even though it is sometimes incipient and difficult to detect. High compression forces operating at this joint may account for the frequency of enthesitis or le- 90 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) sions observed at both the elbow and wrist joints. Microtrauma can provide an opportunity for bacterial involvement leading to inflamation and subsequent ar- thritis. At least some of these osteophytic expressions may be due to one or more of the spondeloarthropathies described in the human medical literature. Collectively, these arthropathies are characterized by inflammatory arthritis, preceding bacterial infection, a strong genetic predisposition, and a propensity for inflammation at the sites where tendons, ligaments, and joint capsules at- tach at the entheses (Dougados et al. 1991 Calin & Taurog 1998). The role that FeSFV plays in the el- evated incidents of AOFs is unknown, but provides at least one link to the presence of polyarthritis in this popu- lation. There was a significant interaction effect between Age Class and Use Area in the severity (and occur- rence) of AOFs, with a greater severity occurring in Use Area 2. This is consistent with earlier studies im- plying that animals in the south were less healthy than in the north, but many management strategies to improve prey base have been implemented. We must consider that we are looking at animals of different ages through a time lens of perhaps 50 years, so it is difficult to pin- point the specific time frame of the interaction. The prevalence of total AOFs prior to 1995 (60.8%) was significantly higher than in animals that were born after 1995 (36.7%). Arthritis was the most common AOF in both the pre- and post-1995 samples. There has been a decrease in the incidence of pathology over time. The presence of many more young animals in the current population (relative to pre-1995 populations) may bias the results. However, we cannot rule out, and we can hope, that animals living today are healthier, less genetically compromised, and therefore less likely to develop severe pathologies. Whatever gains have been made as a result of increased variability through out- breeding or prey species management could be reversed as the increase in panther numbers create greater com- petition for habitat and prey, and offspring continue to mate with each other. Our initial goal of assessing the effect of out-breed- ing on the health of Florida panthers vis-à-vis their oste- ology was limited by sample sizes in the post-1995 higher age classes. There was only one animal in AC-V and nine in AC-IV of the post-1995 sample, compared to 17 and 26, respectively, in pre-1995 sample. Our sample sizes in each group were also skewed with 79 pre-1995 and 49 post-1995. We cannot say with certainty that the out-breeding initiative played an important role in the improved health represented by our results as only eight animals known to be the product of the genetic cross between Texas and Florida cats were included in our study. Most other panthers, with the exception of the Everglades cats, showed at least one of the inbred pan- ther characters of kinked tail or cowlick, and therefore our sample included many more of the original inbreed panther stock. Without the benefit of genetic diversity, we are left with the same basic problems of an inbred population: namely lowered disease resistance and re- duced vitality (Roelke, et al. 1993a,b). We have identified that a predisposition for joint disease may exist in Florida panthers and offer this as- sessment as a first step in developing a protocol to char- acterize the nature of this disease. That bone is limited in the way it can respond to any particular “insult” may limit the ability to distinguish among diseases when study- ing museum skeletons. Therefore, we recommend fol- low-up with studies that include soft tissue analyses, his- tology, bone density tests and radiology of joints includ- ing the forearm and pelvis joints as the latter is often involved in SpA. There is new information revealed by this study and the potential for new paths of discovery. Recom- mendations by a panther advisory commission encour- ages analyses to consider research in toxins, diseases, and panther health, as well as how the prevalence of abnormalities in panthers is correlated with, or interacts with, genetic status (Beier et al. 2003). Our results, together with the rich resource of archival material, leads to new cooperative research opportunities between mu- seums, wildlife biologists, and wildlife veterinarians in the efforts to improve the conservation status of Florida panthers. ACKNOWLEDGEMENTS We would like to thank Geordie Duckler and Blair Van Valkenburgh, University of California, Berkley, for their advice and the use of data and photographs in a Florida panther Symposium that gave rise to this paper. An- thony Falsetti, forensic specialist, along with the staff and students of the C. A. Pound Human Identification Laboratory, University of Florida, provided invaluable assistance with X-ray equipment and forensic informa- tion. Mark Cunningham, Florida panther veterinarian, and Bambi Ferree, and Darrell Land, all of the Florida Fish and Wildlife Conservation Commission, responded to many requests for panther historical and biomedical information, as did Melody Roelke, National Institute of Health. Forensic anthropologist Suzanne Abel, assisted in our understanding of human and animal osteopathology. 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UF ID FP# AGE PRE-POST USE AREA2 TOTAL OSTEO AT4 IN UN GT TOTAL CLASS 19951 AOFs SEVERITY3 HARRIS LINES 9789 3 1 N 2 1 AT1 UN 10424 UCFP 06 3 1 S 1 1 IN 2 11915 2 1 S 0 0 0 11927 UCFP 05 2 1 N 0 0 . 14390 UCFP 02 2 1 S 0 0 . 14699 R. ALLEN 3 1 N 0 0 16374 6 4 1 S 2 2 AT1 GT 1 18798 3 4 1 S 0 0 3 18847 14 5 1 S 1 2 AT2 4 18944 PCO 047 3 1 N 3 2 AT1 UN . 19077 CB 17 3 1 N 0 0 19090 4 1 N 0 0 5 19096 1 5 1 S 4 3 AT1 IN GT 2 20777 2 5 1 S 4 3 AT2 IN GT 0 20957 UCFP 13 2 1 S 0 0 . 20958 UCFP 12 4 1 S 0 0 . 20973 UCFP 14 2 1 S 0 0 22409 7 4 1 S 3 2 AT2 0 22529 4 5 1 S 2 1 AT1 1 23849 UCFP 34 2 2 N 0 0 0 23986 10 2 1 N 5 2 AT1 IN UN 0 24042 Volusia Co.4 1 N 4 3 AT1 2 24096 13 4 1 N 1 1 AT1 0 24160 4 1 S 1 1 GT . 24267 8 5 1 S 1 1 IN 4 24268 PCO 059 4 1 S 1 1 IN . 24314 20 4 1 N 1 2 IN 2 24315 25 4 1 N 1 1 AT 0 24316 24 4 1 N 0 0 3 24557 27 3 1 S 2 3 AT3 GT . 24561 3 1 N 4 3 AT1 UN GT . 24563 15 4 1 S 0 0 3 24595 33 3 1 N 0 0 0 24611 35 1 1 N 0 0 0 24621 30 2 1 S 0 0 0 24644 39 3 1 S 3 1 AT1 UN 0 24645 UCFP 19 1 1 N 0 0 1 24646 17 4 1 N 3 2 AT2 2 24928 18 5 1 N 6 1 AT1 GT 4 24929 41 3 1 N 0 0 1 24931 37 4 1 N 1 1 AT1 . 25908 76 3 2 S 0 0 2 25914 84 2 2 N 0 0 0 25922 PCO 192 1 1 N 0 0 0 26083 43 3 1 N 0 0 1 26157 28 4 1 N 3 2 AT1 UN 0 26159 29 3 1 N 3 1 AT1 96 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) 26161 22 4 1 S 0 0 0 26840 47 2 1 S 0 0 3 26841 44 3 1 S 1 3 GT 0 26842 53 1 1 N 0 0 0 26843 50 3 1 N 1 1 AT2 2 26844 34 4 1 N 1 2 AT1 2 26845 UCFP 21 1 1 N 0 0 . 26856 UCFP 22 3 1 N 0 . 0 26938 UCFP 23 1 1 N 0 0 0 26939 26 4 1 N 2 3 AT3 5 27148 31 5 1 N 2 1 AT1 3 27370 38 4 1 N 4 2 AT1 IN UN 3 27616 12 5 1 N 4 3 AT3 IN GT 2 27618 52 3 1 N 0 0 3 27700 42 4 1 S 5 1 AT1 UN 1 28713 UCFP 30 2 1 S 0 0 4 28802 58 3 1 N 0 0 0 28980 40 5 1 S 2 1 AT1 1 29199 UCFP 25 3 1 N 2 1 AT1 0 29242 UCFP 26 3 1 S 4 2 AT2 GT 3 29250 68 4 1 N 2 2 AT3 IN 5 29262 45 4 1 N 4 2 AT3 2 29263 51 4 1 S 2 2 AT2 GT 2 29273 72 3 1 N 2 3 AT3 GT 1 29370 46 4 1 N 4 3 AT2 UN 3 29532 64 3 2 N 1 3 GT 1 29566 UCFP 33 1 2 N 0 0 5 29567 74 3 2 N 0 0 1 29621 36 5 1 S 1 1 AT1 6 29819 63 4 2 N 3 3 AT1 GT 2 29821 16 5 1 S 6 3 AT3 UN 6 29826 80 4 2 N 1 1 AT1 ̀ 0 30022 UCFP 35 2 2 N 0 0 0 30023 UCFP 36 2 2 N 0 0 0 30064 89 3 2 S 2 3 AT3 2 30178 90 2 2 N 3 2 AT1 GT 4 30366 UCFP 39 1 2 S 0 0 1 30367 UCFP 38 2 2 N 0 0 0 30374 UCFP 43 3 2 N 1 2 GT 0 30391 11 5 1 N 2 1 AT1 3 30393 23 5 1 N 3 3 AT1 IN UN 5 30398 UCFP 40 1 2 S 0 0 3 30399 UCFP 41 2 2 N 0 0 2 30430 UCFP 29 3 1 N 0 0 0 30431 97 2 2 N 0 0 1 30433 UCFP 27 2 2 N 0 0 6 30434 105 4 2 N 3 1 AT1 IN UN 6 30935 49 5 1 N 0 0 5 30936 UCFP 46 1 2 N 0 0 0 30937 UCFP 45 3 2 N 1 3 UN 0 30938 96 2 2 N 0 0 1 30948 98 4 2 N 0 0 2 30957 111 5 1 N 0 0 2 UF ID FP# AGE PRE-POST USE AREA2 TOTAL OSTEO AT4 IN UN GT TOTAL CLASS 19951 AOFs SEVERITY3 HARRIS LINES WILKINS, ALLEN and REED: Florida Panthers 97 30958 92 3 2 N 1 1 AT1 2 30959 UCFP 42 2 2 N 0 0 0 30960 32 5 1 N 2 1 AT1 5 31010 UCFP 52 2 2 N 0 0 0 31011 67 4 2 N 0 0 0 31012 UCFP 48 1 2 N 0 0 0 31018 106 3 2 N 1 1 AT1 2 31019 UCFP 49 2 2 N 0 0 0 31020 108 2 2 N 0 0 2 31021 78 3 2 N 1 1 AT1 3 31022 UCFP 54 1 2 N 0 0 1 31023 UCFP 58 1 2 N 0 0 0 31024 UCFP 50 3 2 N 1 1 AT1 5 31025 UCFP 53 3 2 N 0 0 3 31026 82 4 2 N 0 0 1 31101 91 4 2 S0 0 0 31103 UCFP 66 2 2 S0 . 0 31104 K 94 3 2 S1 1 AT1 5 31106 55 5 2 S4 1 AT1 UN 3 31108 UCFP 62 1 2 S0 0 0 31109 UCFP 65 2 2 N 0 0 4 31110 114 2 2 N 0 0 0 31161 UCFP 69 2 2 S1 1 AT1 0 31162 UCFP 63 3 2 S2 3 AT1 GT 2 31163 59 4 1 N 4 2 AT2 UN 3 31165 115 4 2 N 2 1 AT1 2 31182 69 4 2 N 1 1 AT1 1 31183 109 5 1 N 6 3 AT3 0 1Designates animal born before (1) or after (2) 1995 2Panther used areas to the North or South of Alligator Alley 3Measure of Severity (1) slight (2) moderate (3) represents broken bones, advanced debilitating arthritis, or other extreme condition UF ID FP# AGE PRE-POST USE AREA2 TOTAL OSTEO AT4 IN UN GT TOTAL CLASS 19951 AOFs SEVERITY3 HARRIS LINES 98 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 47(3) 80968 UF MNH cvr.pdf 80968 txt check.pdf