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1 Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA
2 Johne’s Information Center, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706, USA
4 Corresponding author (email: kerri.pedersen{at}aphis.usda.gov)
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Johne’s disease was first identified in an endangered free-ranging Florida Key deer in 1996 at a private residence on Big Pine Key; a second case was confirmed 2 yr later at the same location (Quist et al., 2002). Based on a subsequent survey of repository serum and fecal samples and live capture, the prevalence of Map infection was thought to be low in the Key deer population. However, from 2003 to 2004 five additional deer were diagnosed with Johne’s disease at the same residence and neighboring islands. These reports plus new findings in Johne’s disease research indicating that nonruminant wildlife are also susceptible to infection on heavily contaminated premises (Beard et al., 2001; Corn et al., 2005) raised the possibility that the infection prevalence had increased or was more extensive than previously thought. Additional concerns included illegal feeding of Key deer and the National Key Deer Refuge policy for translocation of deer to keys previously within the historic range of this species. The purpose of this survey was to determine the geographic distribution of Map in the Key deer population.
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MATERIALS AND METHODS |
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The Florida Keys are a series of islands that extend from the southern tip of the Florida peninsula. Key deer occupy several islands along this chain known as the Lower Keys from Little Pine Key to Sugarloaf Key (Hardin et al., 1984). However, approximately 75% of the population is limited to Big Pine and No Name Keys (24°44'N, 81°20'W) where fresh water is available (Lopez, 2001).
Capture methods
Key deer were live-trapped at various locations on Big Pine Key. Locations were selected based on the propensity of Key deer to congregate in the area and the feasibility of setting up a net. A drop net was used to capture the deer according to methods described by Lopez et al. (1998). Once caught in the net, deer were physically restrained while blood and fecal samples were collected. Sex, age, location, and global positioning system coordinates were recorded, and each animal was either marked with tattoo ink or tattooed before release to prevent repeat sampling.
Collection of samples
Samples were collected intermittently from February 2005 through May 2006. Key deer killed by vehicles or other causes were stored in a freezer by National Key Deer Refuge personnel before each survey period, and they were then necropsied as time permitted during the sampling period. Deer killed during the sampling period were necropsied within a few hours of discovery. Fecal pellets and tissue samples including liver, ileum, and mesenteric lymph node were collected from each deer depending on the condition of the animal when found. Blood was collected from freshly killed deer by cardiac puncture; serum was obtained within a few hours of collection and stored in a freezer at –18 C until shipped. Tissue and fecal samples were placed in individual whirl-paks (Fischer Scientific, Suwanee, Georgia, USA). Additional sections of each of the tissues were fixed in 10% buffered formalin, and they were stored for histopathologic evaluation of tissues testing positive for Map. Fecal pellets were collected from the ground at various locations on Big Pine, No Name, Howe, Water, Little Pine, Cudjoe, and Big Torch Keys, and on Munson Island, Little Palm Island, and an unnamed offshore island. Fecal pellets were collected opportunistically in areas where Key deer were known to congregate or where they were observed frequently. The same location was not sampled more than once per week. Only fecal pellets that were fresh as determined by the collector were submitted. Samples were refrigerated for less than or equal to 72 hr and shipped on ice packs to the Johne’s Information Center at the University of Wisconsin (Madison, Wisconsin, USA).
During June and July, raccoons and feral cats were captured on Munson Island and the southern end of Big Pine Key using Tomahawk live-traps. Animals were immobilized with Telazol® (Fort Dodge, Overland Park, Kansas), and then they were euthanized by intracardiac injection of sodium pentobarbital. Necropsies were conducted immediately, and fecal and tissue samples including liver, ileum, and mesenteric lymph node were collected from each animal.
Laboratory methods
Culture, isolate identification, and serology were conducted by the Johne’s Information Center (Madison, Wisconsin, USA). Isolation of mycobacteria was performed using the radiometric method of detection (Collins et al., 1990). Briefly, 3 g of fecal material was processed with a decontamination solution overnight. Then, 10 ml of the supernatant was filtered, and the filter was placed in BACTEC incubation bottles (Bectin Dickerson, Sparks, Maryland) and monitored weekly for 14C release. Aliquots were taken for acid-fast staining from samples signaling positive. Acid-fast organisms isolated from the samples were identified as Map by an IS900 DNA probe and mycobactin-dependent growth patterns. Sera were tested for antibody to Map by a version of an enzyme-linked immunosorbent assay using a protein G antibody conjugate (IDEXX, Portland, Maine, USA; Tryland et al., 2004). Histopathology was conducted at the Southeastern Cooperative Wildlife Disease Study (Athens, Georgia, USA). Tissues from culture-positive animals were embedded in paraffin, and they were sectioned at 3 to 4 µm. Individual sections were stained with hematoxylin and eosin for routine examination and with Ziehl-Neelsen acid-fast stain to search for acid-fast bacteria.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Two of the positive samples on Big Pine Key were collected on the south side of highway US 1, and one sample was from a private residence on Long Beach Road (Fig 1). The only other positive fecal isolate was obtained from a fecal sample collected directly from a clinically ill deer. Samples collected from the other nine keys were negative for Map.
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Two of 97 serum samples tested positive for antibodies to Map. One sample was collected from a deer found dead on Little Palm Island; corresponding fecal and tissue samples tested positive. The other sample was from an adult buck found dead on Big Pine Key but corresponding fecal and tissue samples all tested negative.
Tissue and fecal samples were submitted from 30 raccoons, 3 feral cats, an opossum and a Lower Keys marsh rabbit that had been killed by a vehicle (Table 1). All tissues were culture negative except the mesenteric lymph node of one raccoon captured on Munson Island. No other tissues from this animal were culture positive and histopathology revealed mild inflammation but no acid-fast bacteria typically observed with Map infection. Fecal pellets from three rabbits, two silver rice rats (Oryzomys argentatus), and nine raccoons were collected from the ground but also tested negative (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Key deer comprise most if not all of the ruminants present in the Lower Keys. Although we do not know how or when Map was introduced into the Key deer population, it seems that Map is being maintained in this population. Furthermore, during collection of fecal pellets on Little Palm Island, a Key deer was observed swimming from Little Palm to another island, suggesting that the potential for spread of the infection on a larger scale exists and that it may not remain limited to the area south of US 1. Supplemental feeding, as occurs on Big Pine Key, Munson, and Little Palm islands, encourages congregation of Key deer, which increases animal density, environmental contamination, and the likelihood of transmission of various infectious diseases (Williams, 2001; Nettles et al., 2002). High population density and poor habitat quality as exist in the southern part of Big Pine Key (Harveson et al., 2004) increase the probability of exposure and subsequent infection with Map. The urbanization of the Key deer as described by Folk and Klimstra (1991) encourages free-ranging deer to congregate, which may perpetuate Map in the environment.
Based on the limited number of samples tested, evidence of infection in nonruminant species was scant; a single isolate from the mesenteric lymph node of a raccoon captured on Munson Island was culture positive. Isolation of Map in the tissue indicated that the raccoon had been infected, but neither lesions nor evidence of shedding at the time of capture was found. The raccoon may have become infected through exposure to the contaminated environment (75 [9%] Key deer fecal samples collected on Munson Island [41 ha] were Map positive) or by scavenging an infected deer.
The contribution, if any, of nonruminant wildlife to inter- or intraspecies dissemination of Map is not yet understood. Previous studies have reported infection in raccoons (Corn et al., 2005) as well as other nonruminant species that inhabit dairy farms with infected livestock, including feral cats (Palmer et al., 2005), rabbits (Raizman et al., 2005), birds (Corn et al., 2005), and coyotes (Anderson et al., 2007). Many of these studies describe clinically and histopathologically normal animals from which the isolation of Map from tissue seems to have been an incidental finding. It is possible that some nonruminant species are not affected by Map infection or that these animals may have been tested during the early stages of infection before lesion development. In Scotland, Map seems to be established in rabbits due to high Map excretion rate and the grazing habits of rabbits along with horizontal, vertical and pseudovertical transmission (Judge et al., 2006). A more recent study detected an unusually high infection rate (81 animals; 38%) in raccoons and other scavenging animals in Wisconsin (Anderson et al., 2007); this rate was based on the results of three different Map polymerase chain reaction assays.
Key deer mortality caused by Map infection is relatively low in comparison to vehicle-related mortality (2002–2004; 72%). However, due to the endangered status of the Key deer and the unknown factors affecting the perpetuation and dispersal of the microorganism in the Lower Florida Keys, it is imperative to minimize the risk of infection. Actions that may reduce risk include 1) increased education of tourists and residents about the consequences of supplemental feeding (Lopez et al., 2003), 2) increased enforcement of laws prohibiting illegal feeding of deer (Miller et al., 2003), and 3) continued monitoring of the Key deer population to determine whether the disease continues to be maintained and whether dissemination to areas north of US 1 occurs. Further studies on the role of environmental contamination in the maintenance and transmission of Map and studies on the effects of cessation of supplemental feeding of Key deer on dispersal are recommended.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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LITERATURE CITED |
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BEARD, P. M., M. J. DANIELS, D. HENDERSON, A. PIRIE, K. RUDGE, D. BUXTON, S. RHIND, A. GREIG, M. R. HUTCHINGS, I. MCKENDRICK, K. STEVENSON, AND J. M. SHARP. 2001. Paratuberculosis infection of nonruminant wildlife in Scotland. Journal of Clinical Microbiology 39: 1517–1521.
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Received for publication 1 August 2007.
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