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1 Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine;
2 D. B. Warnell School of Forest Resources, The University of Georgia, Athens, Georgia 30602, USA;
3 Johne’s Testing Center, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706, USA
5 Corresponding author (email: rdavidso{at}vet.uga.edu)
ABSTRACT:
From July 1998 through October 2002, radiometric culture (ileocecal lymph node, mesenteric lymph node, and feces) and serologic testing by enzyme-linked immunosorbent assay (ELISA) were used to survey white-tailed deer (Odocoileus virginianus) from the southeastern United States for infection by Mycobacterium avium subsp. paratuberculosis (Mptb), the causative agent of paratuberculosis (Johne’s disease). Mycobacterium avium subsp. paratuberculosis was isolated from the ileocecal lymph node of one of 313 deer (0.3%) originating from 63 populations in Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, South Carolina, Tennessee, and West Virginia (USA). Six deer (2%), all from different populations, had ELISA results above a 0.25 sample-to-positive cutoff value, but none of the ELISA reactors originated from the population from which the single Mptb isolation was made. These six deer were seronegative when tested by agar gel immunodiffusion (AGID). Collectively, these data indicate that white-tailed deer currently do not constitute a broad regional reservoir for Mptb; however, further study is warranted to clarify the significance, if any, of infected deer to the epizootiology of paratuberculosis on a local scale. Adaptation and validation of an ELISA or another serologic assay for use with deer and other wildlife would markedly enhance Mptb surveillance among wild populations and would be a powerful tool for gaining information on the role of wild species in epidemiology of paratuberculosis.
Key words: ELISA, Johne’s disease, Mycobacterium avium subsp. paratuberculosis, Odocoileus virginianus, radiometric culture, survey, United States, white-tailed deer.
Paratuberculosis, or Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (Mptb), occurs worldwide and is recognized as a significant health problem for domesticated ruminants, wild ruminants in zoologic collections, and farmed cervids (Williams, 2001). In the United States, a 1996 national survey found that 22% of dairy cattle herds had at least a 10% infection rate and 41% of herds had at least one cow positive by enzyme-linked immunosorbent assay (ELISA) (Wells and Wagner, 2000). Infection with Mptb has been confirmed in multiple farmed red deer (Cervus elaphus elaphus) herds in the United States and in more than 300 red deer operations in New Zealand (Mackintosh, 2002).
Compared with traditional livestock or captive wild ruminants, reports of clinical or subclinical infection with Mptb among free-ranging wild ruminants are infrequent (Williams, 2001; Quist et al., 2002). Among free-ranging native wild ruminants in North America, clinical paratuberculosis has been reported in populations of Rocky Mountain bighorn sheep (Ovis canadensis; Williams et al., 1979), tule elk (C. elaphus nannodes; Jessup et al., 1981; Cook et al., 1997; Manning et al., 2003), Rocky Mountain elk (C. elaphus nelsoni; Manning, un-publ. data), and Key deer (Odocoileus virginianus clavium; Quist et al., 2002). Additionally, Mptb was isolated from two of 10 clinically normal wild white-tailed deer from a cattle farm of known infection (Chiodini and Van Kruinningen, 1983). Both mule deer (Odocoileus hemionus) and white-tailed deer have been infected experimentally (Williams et al., 1983a, b). Worldwide, Mptb infection has been infrequently reported in nonruminant species as well, including, for example, primates (Zwick et al., 2002) and rabbits (Beard et al., 2001).
Although it is clear that free-ranging wild ruminants are susceptible to infection, in North America, the infection apparently is maintained within only a few wild populations (Williams, 2001). A better understanding of the prevalence, distribution, and ecology of paratuberculosis within wild ruminant populations is desirable from a wildlife health perspective. Furthermore, this epidemiologic information would be important in the development of a national paratuberculosis control program for livestock. Here, we report a culture and serologic survey for Mptb infection among white-tailed deer populations in the southeastern United States.
White-tailed deer in this study were collected for population health evaluations by the Southeastern Cooperative Wildlife Disease Study (SCWDS, Athens, Georgia, USA) on the request of state or federal natural resource management agencies from July 1998 through October 2002. These health evaluations involved necropsy and laboratory testing of five adult animals from each of 63 deer populations in Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, South Carolina, Tennessee, and West Virginia (USA). Two exceptions were six deer tested from one population in Mississippi and two deer tested at one location in South Carolina. Animals were collected by cervical gunshot, temporarily chilled for 6–12 hr, and necropsied within 8–18 hr of death. At necropsy, the ileocecal lymph node, an approximately 3-cm segment of mesenteric lymph node, and about 10–25 g of feces per rectum were collected, placed in separate plastic bags (Whirl-Pak®, Fisher Scientific, Suwanee, Georgia, USA), stored on cold packs, and shipped by overnight courier to the Johne’s Testing Center (School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin, USA). Culture for Mptb was conducted on each of these samples with a radiometric (BACTEC) technique, and the identities of mycobacterial isolates were confirmed by IS900 DNA polymerase chain reaction probe, mycobactin-dependent growth patterns, and high-performance liquid chromatography (HPLC) analyses of mycobacterial mycolic acids. Culture and identification methods used in this study previously have been described in detail (Collins et al., 1990; Quist et al., 2002). Acid-fast isolates determined not to be Mptb according to morphology, lack of mycobactin dependency, and absence of IS900 were identified by HPLC methods (Butler et al., 1991).
Immediately following death, blood was collected from each animal by cardiac puncture, placed in a 50-ml tube and allowed to clot; serum was harvested within 12 hr and stored at –20 C for serologic testing. Sera were tested by a commercially available ELISA assay (IDEXX, Portland, Maine, USA). As per kit instructions for cattle and in accord with a previous study of Key deer (Quist et al., 2002), an ELISA sample-to-positive (S/P) value of 0.25 was chosen as the cutoff value to discriminate test-positive sera from test-negative sera, although the S/P value yielding the best sensitivity and specificity rates for this assay in white-tailed deer is not known. Sera with S/P>0.25 were also tested by agar gel immunodiffusion (AGID; Immunocell, Portland, Maine, USA).
Cultures were successfully completed on 930 of 939 (99%) samples from 313 deer; nine samples (1%) were lost to contamination. All three samples were completed for 306 deer; among the remaining seven deer, cultures were successfully completed on both lymph nodes for three deer, one lymph node and feces for two deer, and feces only for two deer. Mycobacterium avium subsp. paratuberculosis was isolated from the ileocecal lymph node of one deer from Kentucky, from which all three samples were cultured to completion (Table 1
). Two other species of mycobacteria—one isolate of Mycobacterium abscessus and two isolates of Mycobacterium terrae complex—were obtained from deer originating in Arkansas, Tennessee, and West Virginia. Mycobacteria were isolated from a single sample for each of these four deer, and gross lesions suggestive of mycobacterial infection were not noted in any of the deer from which mycobacterial isolates were obtained.
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0.25 by ELISA (Fig. 1), whereas the remaining six deer (2%) had S/P values above 0.25 (viz., 0.34, 0.39, 0.39, 0.44, 0.83, 1.65). The six ELISA-reactive deer were from five populations, but none were from the population from which the Mptb isolate was obtained, including the culture-positive animal. All six of the samples with ELISA values above 0.25 were negative when tested by AGID. The negative AGID results might indicate that the antibody detected by the ELISA was elicited by an infection with a pathogen other than Mptb because AGID, although considered less sensitive, is regarded as a more specific assay in some species (Shulaw et al., 1993). The overall ELISA results suggest a low prevalence of any mycobacterial infections among the deer examined.
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The single culture-positive deer was collected in August 2000 from Kleber Wildlife Management Area (WMA, Owen County, Kentucky), but whether this culture-positive deer reflected independent maintenance of Mptb within wild deer or acquisition from a domestic animal source is unknown. In each prior instance in which Mptb infection was confirmed among North American wild ruminant populations, potential risk factors were described, including high population density (Cook et al., 1997; Jessup and Williams, 1999; Quist et al., 2002), gregarious behavior and repeated use of traditional bedding areas (Jessup and Williams, 1999), concentration at supplemental feeding sites (Quist et al., 2002), and presence of infected cattle (Chiodini and Van Kruinningen, 1983). A portion of Kleber WMA had been a dairy farm prior to its acquisition by the Kentucky Department of Wildlife and Fisheries in 1962, and dairy and beef cattle farms currently exist in the immediate vicinity of Kleber WMA. Of the risk factors noted above, the only one potentially present at Kleber WMA is a domestic ruminant source. In the two prior instances of confirmed paratuberculosis among wild white-tailed deer, cattle were a probable source for a local Connecticut deer population (Chiodini and Van Kruinningen, 1983), but a domestic animal or other extrinsic source was not identified for an insular Key deer population (Quist et al., 2002).
Although culture surveys to estimate infection rates have been conducted on a few local wild ruminant populations (bighorn sheep, tule elk, and Key deer) known or suspected to independently sustain Mptb infection (Williams et al., 1979; Cook et al., 1997; Quist et al., 2002), larger scale surveys for Mptb are lacking for other native North American wild ruminants. As potential for exposure among wildlife increases because of shared range or contact with infected domestic species, it will become important to monitor the spread, if any, of the infection into wildlife and the possibility of its return to domestic species (Daniels et al., 2003; Deutz et al., 2003).
When choosing population screening methods, one must take into account expected prevalence, sample collection and processing requirements, the sensitivity of the assays, and cost. Detection of Mptb infection in a single survey for low-prevalence herds is difficult by any method, and serial testing with multiple assays might be necessary to obtain the most accurate assessment of prevalence. Culture, microscopic, or both types of examination of extraintestinal tissue is a critical component of surveillance and is necessary to rule out the possibility that an isolation of Mptb from a fecal sample was a result of "pass-through" from a contaminated environment vs. true infection. Preferred samples for isolation are mesenteric and ileocecal lymph nodes and ileum. Assays of tissues from animals with clinical signs compatible with Johne’s disease (such as poor body condition or diarrhea) are likely to be more rewarding than from apparently healthy animals.
Fecal sample pooling can be used to reduce the overall cost of culture surveillance but is less sensitive than individual sample testing (Sergeant et al., 2002; Wells et al., 2002; Schroen et al., 2003). The sensitivity of pooled fecal samples specifically in free-ranging wildlife has not been evaluated. Freezing of samples should be avoided to maintain the viability of as many organisms as possible (Richards, 1977).
Serum is a standard sample for wildlife health surveillance (assays can be performed for antibodies to many pathogens, storage is possible for long periods with minimal degradation of the antibody, and rarely is contamination at collection a problem). Testing by ELISA can be a fast and inexpensive first-step method to "triage" wildlife populations into probable infected vs. uninfected categories; however, the current ELISA has not been fully validated in deer, making interpretation of results somewhat speculative (Shulaw et al., 1986; Williams, 2001; Quist et al., 2002). An ELISA with a cervid-specific conjugate or protein G conjugate should be used for populations of deer, and interpretation should be done on a herd rather than on an individual animal basis (Collins, 1996; Kramsky et al., 2003). Because of its low sensitivity, the AGID is not recommended for use with deer (Schroen et al., 2003). Ultimately, an array of validated assays potentially could establish Mptb status for populations as a whole, indicate the probable prevalence of infection within populations, and provide the basis for confirmatory culture-based surveillance to those populations with the greatest likelihood of Mptb infection.
Support for this work was provided in part through sponsorship of SCWDS by the fish and wildlife agencies of Alabama, Arkansas, Florida, Georgia, Kansas, Kentucky, Louisiana, Maryland, Mississippi, Missouri, North Carolina, Puerto Rico, South Carolina, Tennessee, Virginia, and West Virginia. Funds were provided by the Federal Aid in Wildlife Restoration Act (50 Stat. 917). Additional support was provided through Cooperative Agreements 1998, 1999, 2000, 2001, and 2002-9613-0032-CA (Veterinary Services, Animal and Plant Health Inspection Service, US Department of Agriculture) and through Grant Agreements 1445-GT09-96-0002 and 01ERAG0013 (Biological Resources Division, US Geological Survey, US Department of the Interior [USDI]). We thank numerous personnel with SCWDS-sponsoring state wildlife agencies, the Division of Wildlife Refuges (US Fish and Wildlife Service, USDI), and the National Park Service (USDI) for assistance with logistical and field aspects of SCWDS deer health evaluation activities. Sincere appreciation also is extended to many current and former SCWDS staff for their exceptional technical assistance, most notably D. M. Kavanaugh, M. P. Luttrell, L. A. Lewis-Weis, and M. A. Stevenson.
4 Current address: Wildlife Health Associates Inc., PO Box 109, Dillon, Montana 59725, USA 
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Received for publication 21 May 2003.
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