Abstract
Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants (“7,5”) appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock.
Mycobacterium avium subsp. paratuberculosis infection can be an economically disruptive herd health problem for domestic ruminant species. The infection results in Johne's disease, a chronic granulomatous gastroenteritis leading to emaciation and death. Cases of the infection also have been reported in a number of free-ranging ruminant species (28). Elk (Cervus elaphus) at Point Reyes National Seashore in California (16) and a population of bighorn sheep (Ovis canadensis) in Colorado have remained infected over several years (13). Ongoing studies in The Netherlands and Italy suggest the involvement of roe deer (Capreolus capreolus) in the epidemiology of M. avium subsp. paratuberculosis in isolated areas with associated livestock infection (20, 24, 26).
There are few reports of M. avium subsp. paratuberculosis isolations from nonruminant free-ranging wildlife. A suspected isolate of M. avium subsp. paratuberculosis was reported from a European brown hare (Lepus europaeus) in England in 1977 (17), and lesions consistent with M. avium subsp. paratuberculosis infection were described in a rabbit (Oryctolagus cuniculus) in Scotland in 1990 (2). The initial confirmation of M. avium subsp. paratuberculosis in free-ranging nonruminant wildlife was from rabbits on farms with a history of M. avium subsp. paratuberculosis infection in Scotland (5, 11, 12). Subsequently, M. avium subsp. paratuberculosis was isolated from tissue samples from foxes (Vulpes vulpes), stoats (Mustela erminea) (3), weasels (Mustela nivalis), badgers (Meles meles), wood mice (Apodemus sylvaticus), Norway rats (Rattus norvegicus), European brown hares, jackdaws (Corvus monedula), rooks (Corvus frugilegus), and crows (Corvus corone) (4) in these same areas. A study based on rabbit and deer fecal samples collected from the ground on dairy farms with infected cattle in Minnesota yielded M. avium subsp. paratuberculosis isolates from two eastern cottontail fecal samples and two white-tailed deer fecal samples (23).
For the most effective control of this challenging disease in domestic livestock and to assess its threat, if any, to wildlife health, it is important to determine the extent to which wildlife species may serve as reservoirs for the organism. In its report on Johne's disease, the National Research Council recommended that field studies be conducted to assess natural infections of wildlife near livestock operations with endemic Johne's disease (19). The goal of the present study was to evaluate both the presence of M. avium subsp. paratuberculosis in tissue and M. avium subsp. paratuberculosis shedding by free-ranging mammals and birds sharing habitat with infected livestock in two ecologically different regions of the United States.
MATERIALS AND METHODS
Study sites.
Intensive sampling of wildlife was conducted on nine livestock premises. Study sites were chosen in two regions to evaluate wildlife and wildlife-livestock interactions under the different environmental and ecological conditions. In Wisconsin, specimens were collected from four dairy farms known to contain livestock infected with M. avium subsp. paratuberculosis (Farm W1, Monroe County; W2, Outagamie County; W3, Sheboygan County; W4, Jackson County). In Georgia, specimens were collected from two dairy farms (G2, Brooks County; G3, Jefferson County) and three beef cattle farms (G1, Early County; G4, Oglethorpe County; G5, Grady County) with histories of M. avium subsp. paratuberculosis-infected livestock. All samples collected were from free-ranging animals captured on the selected premises and, in the case of the dairy farms, in the immediate vicinity of the dairy barns.
The infection prevalence in cattle for Wisconsin premises was determined by consecutive years of fecal culture and serologic testing. Prevalence data for G1 came from its long-term participation in a U.S. Department of Agriculture, Agricultural Research Service project on M. avium subsp. paratuberculosis. Fecal cultures were used to determine the prevalence for Georgia premises G2-G5. Farms W4 and G5 initially were considered to be negative controls: all cows on the farms had tested negative by enzyme-linked immunosorbent assay or fecal culture prior to our survey. However, one W4 cow and six G5 cows were confirmed to be infected with M. avium subsp. paratuberculosis after the wildlife sampling had been conducted. These farms were therefore recategorized as infected premises.
Wildlife species evaluated.
Wild mammal and bird species sampled were those with the highest potential for exposure to contaminated materials and/or that posed the highest risk for contamination of livestock feed or forage on the selected farms. Targeted species included mammals of the orders Artiodactyla, Carnivora, Didelphimorphia, Lagomorpha, Rodentia, and Xenarthra. Targeted avian species included members of the order Passeriformes (Columbidae, Corvidae, Emberizidae, Fringillidae, Passeridae, and Sturnidae). Wild animals sampled represented 25 species of mammals and 22 species of birds and were collected by using authorized, species-appropriate methods (live-capture traps and shooting). Methods for capture of birds included mist nets and shooting.
Samples.
Four samples (ileum, liver, intestinal lymph node, and feces) were harvested from the larger mammals. Additional sections of these samples from the larger mammals were fixed in 10% buffered formalin and stored for future histopathological evaluation if M. avium subsp. paratuberculosis was isolated from tissue or feces. Two samples (liver and the entire gastrointestinal tract) were collected from smaller mammals and birds: the gastrointestinal tract was homogenized, and three aliquots were taken from the homogenate for culture. The samples were chilled and shipped overnight to the Johne's Testing Center, University of Wisconsin for culture.
Study size.
Samples were collected from free-ranging wildlife associated with nine cattle premises in Wisconsin and Georgia from September 2003 to January 2004. Attempts were made to acquire samples from the same species at each site to facilitate comparison without introducing species-specific epidemiological variables. The actual number of animals sampled per species was affected by species abundance at the study sites. Observations and capture success data gathered while collections were in progress were used to determine the numbers of individuals to sample per species.
Assays.
Cultures and isolate identification were performed at the Johne's Testing Center by using the radiometric (BACTEC) culture method (6). Acid-fast isolates were identified as M. avium subsp. paratuberculosis using 16S/IS900/IS1311 plus L1/L9 integration site PCRs, amplification of a M. avium subsp. paratuberculosis unique sequence (locus 251) plus mycobactin dependency growth characteristics. Twenty-nine isolates confirmed as M. avium subsp. paratuberculosis were further genotyped by PCR-sequencing of two short sequence repeat (SSR) loci. Cluster analysis and genotype association dendrogram development based on these SSR (G and GGT residue repeats) alleles was performed (1, 15).
Histopathology.
Tissues from culture-positive animals were embedded in paraffin and 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. Histopathology analyses were conducted at the Southeastern Cooperative Wildlife Disease Study.
RESULTS
M. avium subsp. paratuberculosis was isolated from 39 of 2,752 samples (1.4%) collected from 30 of 774 free-ranging animals (3.4%) (Table 1). Culture-positive samples came from 12 free-ranging species, including nine mammalian species (raccoon [Procyon lotor], n = 8; nine-banded armadillo [Dasypus novemcinctus], n = 4; feral cat [Felis catus], n = 2; Eastern cottontail [Sylvilagus floridanus], n = 1; opossum [Didelphis virginiana], n = 1; hispid cotton rat [Sigmodon hispidus], n = 1; Norway rat, n = 1; northern short-tailed shrew [Blarina brevicauda], n = 1; striped skunk [Mephitis mephitis], n = 1) and three avian species (European starling [Sturnus vulgaris], n = 7; house sparrow [Passer domesticus], n = 1; common snipe [Gallinago gallinago], n = 1).
TABLE 1.
Thirty-nine M. avium subsp. paratuberculosis wildlife isolates by site, species, and sample type
| Site | Species | Sample type(s)a
|
||||
|---|---|---|---|---|---|---|
| Ileum | MLN | Liver | Fecal | GI tract | ||
| G1 | Raccoon | + | − | − | − | na |
| Cottontail | + | − | − | − | na | |
| Armadillo | + | + | + | − | na | |
| Cotton rat | na | na | na | na | + | |
| Opossum | − | + | − | − | na | |
| Opossum | − | − | − | + | na | |
| G2 | Raccoon | − | + | − | − | na |
| G5 | Armadillo | − | − | − | + | na |
| Armadillo | − | − | − | + | na | |
| W1 | Shrew | − | − | + | − | na |
| Sparrow | na | na | na | na | + | |
| Feral catb | + | + | − | + | na | |
| Snipe | na | na | na | na | + | |
| W2 | Norway rat | na | na | na | na | + |
| Starling | na | na | − | na | + | |
| Starling | na | na | + | na | − | |
| Starling | na | na | + | na | − | |
| Starling | na | na | − | na | + | |
| Feral cat | + | + | − | − | na | |
| Raccoon | − | + | − | − | na | |
| Raccoon | + | + | − | − | na | |
| Raccoon | − | − | − | + | na | |
| Raccoon | − | + | − | − | na | |
| W3 | Raccoon | − | + | − | + | na |
| Starling | na | na | − | na | + | |
| Starling | na | na | − | na | + | |
| Starling | na | na | − | na | + | |
| W4 | Raccoon | + | + | − | + | na |
| Pocket gopher | na | na | na | na | + | |
| Skunk | − | + | − | − | na | |
| Study totals | 7 | 11 | 4 | 7 | 10 | |
The full gastrointestinal (GI) tract was homogenized, and three aliquots were incubated for small mammals or birds. In some cases, separate liver samples were collected (e.g., starling). Specimens were collected either as pooled gastrointestinal tract (ileum, mesenteric lymph node [MLN], liver, and feces not collected separately [na]) or as the individual tissues and feces (gastrointestinal tract not pooled [na]).
Two lymph node samples were incubated, and isolates were obtained from both.
Shedding, as indicated by the presence of M. avium subsp. paratuberculosis in fecal samples, was detected in seven (0.9%) animals: three raccoons (WI), two armadillos (GA), one opossum (GA), and one feral cat (WI) (Table 2). Isolations of M. avium subsp. paratuberculosis were made from tissue samples from two of these raccoons and the feral cat, but for one raccoon, the two armadillos and the opossum, feces was the sole culture-positive sample. Isolations of M. avium subsp. paratuberculosis were made at least four times from each of the tissue types selected for testing in this survey (Table 2).
TABLE 2.
Total number of infected animals by species
| Species | No. of animals
|
Prevalence (%) | |
|---|---|---|---|
| Infected | Sampled | ||
| Armadillo | 4 | 23 | 17.4 |
| Feral cat | 2 | 18 | 11.1 |
| Hispid cotton rat | 1 | 41 | 2.4 |
| Eastern cottontail | 1 | 56 | 1.8 |
| Opossum | 2 | 54 | 3.7 |
| Raccoon | 8 | 42 | 19.0 |
| Norway rat | 1 | 4 | 25.0 |
| Southeastern shrew | 1 | 4 | 25.0 |
| Striped skunk | 1 | 10 | 10.0 |
| Common snipe | 1 | 1 | 100 |
| House sparrow | 1 | 60 | 1.7 |
| European starling | 7 | 40 | 17.5 |
Acid-fast and hematoxylin-and-eosin-stained tissues from culture-positive raccoons (n = 10), opossums (n = 3), armadillos (n = 3), feral cats (n = 2), and skunk (n = 1) were examined. Neither acid-fast bacteria nor granulomatous infiltrates consistent with M. avium subsp. paratuberculosis infection in ruminants were apparent in any of the tissues examined.
The prevalence of M. avium subsp. paratuberculosis-infected wildlife (number of animals from which M. avium subsp. paratuberculosis was isolated/number animals sampled) ranged from 2.7 to 8.3% on the premises in Wisconsin (Table 3) and from 0 to 6.0% in Georgia (Table 4). Overgrowth of nonmycobacterial organisms in tissue samples may have hindered isolation of mycobacteria at some farms (premises sample contamination ranged from 0.7 to 11%). In the majority of cases, the contaminated sample was the homogenized gastrointestinal tract.
TABLE 3.
M. avium subsp. paratuberculosis isolated from free-ranging wildlife in Wisconsin
| Parameter | Farm
|
|||
|---|---|---|---|---|
| W1 | W2 | W3 | W4 | |
| Farm type/herd size | Dairy/70 | Dairy/1,400 | Dairy/396 | Dairy/50 |
| Herd prevalencea (%) | 17.7 | 10.3 | 13.5 | 2.0 |
| No. of wild animals sampled | 77 | 120 | 101 | 109 |
| No. of samples collected | 323 | 462 | 370 | 367 |
| % Contaminatedb | 5.3 | 8.7 | 8.1 | 0.8 |
| No. of positive samplesd | 6 | 12 | 5 | 5 |
| No. of positive animalsd | 4 | 10 | 4 | 3 |
| Infection prevalence (%) | 5.2 | 8.3 | 4.0 | 2.7 |
| No. of animals sheddingc | 1 | 1 | 1 | 1 |
By ELISA.
Lost due to overgrowth of nonmycobacterial organisms.
M. avium subsp. paratuberculosis isolated from fecal samples.
Positive, that is, for M. avium subsp. paratuberculosis.
TABLE 4.
M. avium subsp. paratuberculosis isolated from free-ranging wildlife in Georgia
| Parameter | Farm
|
||||
|---|---|---|---|---|---|
| G1 | G2 | G3 | G4 | G5 | |
| Farm type/herd size | Beef/740 | Dairy/430 | Dairy/140 | Beef/21 | Beef/69 |
| Herd prevalencea (%) | 1.6 | 7.4 | 0.0 | 9.1 | 10.1 |
| No. of wild animals sampled | 100 | 48 | 63 | 56 | 100 |
| No. of samples collected | 268 | 198 | 240 | 142 | 382 |
| % Contaminatedb | 0.7 | 6.1 | 4.6 | 3.5 | 11.0 |
| No. of samples positived | 8 | 1 | 0 | 0 | 2 |
| No. of animals positived | 6 | 1 | 0 | 0 | 2 |
| Infection prevalence (%) | 6.0 | 2.1 | 0 | 0 | 2.0 |
| No. of animals sheddingc | 1 | 0 | 0 | 0 | 2 |
G1 values were determined by ELISA; G3 to G5 values were determined by individual fecal culture; G2 values were determined by fecal pools of five. A value of 7.4% represents eight positive pools of five cows from a total of 108 pools and assumes one positive cow per pool.
Lost due to overgrowth of nonmycobacterial organisms.
M. avium subsp. paratuberculosis isolated from fecal samples.
See Table 3, footnote d.
There was no statistical correlation between the test-prevalence of M. avium subsp. paratuberculosis in livestock versus wildlife on the farms (r = 0.31). There was no difference in prevalence of M. avium subsp. paratuberculosis infection in wildlife among all farms in general (χ2 = 14.43, df = 8, P > 0.10), but test-positive wildlife were slightly more prevalent on farms in Wisconsin than in Georgia (χ2 = 3.10, df = 1, P < 0.10). Also, there was no difference in prevalence in wildlife on dairy farms versus beef cattle farms (χ2 = 0.32, df = 1, P > 0.10). At least one M. avium subsp. paratuberculosis-infected free-ranging animal was found on all four Wisconsin sites and on three of five Georgia sites. Of the two farms in Georgia where M. avium subsp. paratuberculosis was not isolated from wildlife, one was a dairy with a prior history of Johne's disease at a low level but with test-negative cattle at the time of this survey, the other was a beef operation with a cattle test-positive prevalence of 9.1%.
Twenty-nine of the 39 M. avium subsp. paratuberculosis wildlife isolates were subjected to genotyping based on a recently described set of SSR markers (1, 10, 18). This analysis revealed a total of 10 distinct alleles (Table 5) in 11 animal species. Cluster analysis identified one major allele defined by the G-residue and GGT-residue repeat numbers as “7,5”. This allele was identified in 29% of the 29 M. avium subsp. paratuberculosis isolates studied and was one of two alleles shared among isolates from both geographic locations (Table 5). Georgia isolates had five alleles: “7,5”, “7,3”, “7,4”, “12,5”; and “8,4”. Isolates from Wisconsin carried 9 distinct alleles (Table 5). The raccoon isolates (all from Wisconsin) carried 4 alleles: “14,5”, “7,4”, “7,5”, and “13,5”. Isolates from starlings showed five alleles. Unusually, one M. avium subsp. paratuberculosis isolate from a skunk mesenteric lymph node carried a genotype typical for Mycobacterium avium subsp. avium (identified as 2GC4G, 3GGT). Three isolates from an armadillo carried three different alleles and were distinct from all other M. avium subsp. paratuberculosis isolates in that they carried a unique IS1311-PCR-RFLP profile (data not shown). Similarly, two isolates from a cat showed 2 distinct alleles (“7,5” and “14,3”).
TABLE 5.
Wildlife M. avium subsp. paratuberculosis isolates by short-sequence repeat genotype
| G-GGT pattern | Species (farm of origin) |
|---|---|
| 13G-5GGT | Starling (W2), raccoon (W4) |
| 12G-5GGT | Norway rat (W2)a, starling (W3) |
| 15G-5GGT | Shrew (W1), starling (W2 and W2) |
| 14G-5GGT | Cat (W3, W3, and W3), raccoon (W2), starling (W3), snipe (W1) |
| 7G-5GGT/10G | Cat (W2), raccoon (W2, W3, and W4), starling (W3), opossum (G1), cotton rat (G1), cow (G2) |
| 14G-3GGT | Cat (W2) |
| 2GC4G-3GGT | Skunk (W4) |
| 7G-3GGT | Armadillo (G1) |
| 7G-6GGT | Starling (W2) |
| 7G-4GGT | Cat (W2), raccoon (G2), armadillo (G1) |
| 8G-4GGT | Armadillo (G1) |
No GGT data for this isolate.
DISCUSSION
This report provides evidence of M. avium subsp. paratuberculosis infection in a wide range of nonruminant wildlife species in North America and confirms that the nonruminant infections reported in Scotland (3, 4) were not an isolated occurrence. In both of these studies M. avium subsp. paratuberculosis was isolated from tissue and feces from mammals and birds, and infections were detected on numerous premises. However, both the number of isolates and species infection prevalences were lower in our surveys than reported in Scotland (4, 11, 12).
It is now clear that M. avium subsp. paratuberculosis may infect animals in multiple taxonomic groups, but the pathogenic capacity of the organism for these species is still an open question. In Scotland, histological lesions were seen in rabbits (11, 12), foxes, weasels, a stoat, wood mouse, and crow (4). The absence of histological lesions in tissues from infected animals in our study may be related to the phase of infection in the culture-positive animals examined, to a lack of pathology associated with M. avium subsp. paratuberculosis infection in these species, or to the M. avium subsp. paratuberculosis strain present on these farms. The impact on wildlife health of this organism has yet to be determined.
The isolation of M. avium subsp. paratuberculosis from both tissue and fecal samples in our study is evidence of true infection, not “pass-through” of recently ingested contaminated material. Shedding of M. avium subsp. paratuberculosis may result in wildlife to wildlife, wildlife to livestock, and/or livestock to wildlife transmission. It has been postulated that livestock could become infected by ingesting forage contaminated by infected rabbits as they may excrete up to 4 × 106 CFU per g of feces (7, 8, 9). Although we did not quantify M. avium subsp. paratuberculosis in feces, our detection of shedding in raccoons, opossums, an armadillo and a feral cat is in line with surveys in Scotland in which M. avium subsp. paratuberculosis was found in the feces of foxes (3 of 27), a stoat (1 of 6), wood mice (2 of 2), crows (4 of 12), and a rook (1 of 1) (4).
Fecal contamination of the environment on dairy farms by M. avium subsp. paratuberculosis-infected cows is extensive (22). Given the number of infected cattle in a herd, typical farm animal density and the volume of contaminated feces produced by infected cows, the potential for farm contamination by cattle is many orders of magnitude greater than contamination that would be produced by infected wildlife at the prevalences shown in the present study.
Strain typing using two highly polymorphic SSR loci identified 10 alleles with a predominance of one strain appearing in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Although there are a few “host-associated” M. avium subsp. paratuberculosis alleles, this “7,5” allele is present in cattle, sheep, and goat populations (10). In fact, the“7,5” allele represents ca. 10% of the strains analyzed on a nationwide collection of strains (N. B. Harris, unpublished data). Taken together, the distribution of a major set of alleles and multiple unique genotypes are suggestive of interspecies transmission. The observation that multiple strains were isolated from an armadillo and a feral cat is consistent with several ongoing investigations that have identified multiple alleles in cattle from infected herds (Harris, unpublished). It is likely that a scavenging wildlife species or even cattle in infected herds may carry M. avium subsp. paratuberculosis with different alleles if they are exposed to multiple strains at multiple time points. The significance of this observation in the transmission of M. avium subsp. paratuberculosis within and between herds and the ability of specific M. avium subsp. paratuberculosis clones to predominate in the transmission cycle across species needs to be elucidated.
Spillover of infectious organisms from closely held domestic herds to wildlife is of concern to wildlife managers and livestock producers both because it affects wildlife health and because wild animals may serve as potential reservoirs or disseminators of infection to livestock. A recent study reported that verocytotoxin-producing Escherichia coli isolates obtained from a starling and a Norway rat were identical to cattle isolates on surrounding farms (21). Ruminant and nonruminant wildlife may become exposed to M. avium subsp. paratuberculosis via feeding on contaminated grain, forage in pastures, feces, or on infected prey (8). Given the higher prevalence of M. avium subsp. paratuberculosis-infected cattle on the farms in Wisconsin, the higher prevalence of M. avium subsp. paratuberculosis in wildlife in Wisconsin was predictable. However, there was no correlation between infection prevalence in cattle and wildlife among the nine farms. Factors affecting wildlife exposure to M. avium subsp. paratuberculosis may include microhabitat ecology, organism survival, water and soil conditions, herd size, husbandry methods, and variations in species interactions, plus grooming and feeding habits. No conclusions could be drawn as to a premise's domestic agriculture infection prevalence and surrounding wildlife infection rates in the current study.
Potential routes of transmission of M. avium subsp. paratuberculosis from wildlife back to livestock include fecal contamination of feed and forage, both in farm buildings and in pastures (8). Individuals of some wildlife species may live for several years with home ranges that cover areas large enough to include more than one farm. Raccoon home ranges average 40 to 100 ha, and movements may be made to areas outside the home range to visit temporary food sources (14). Infected wildlife may shed organisms such as M. avium subsp. paratuberculosis over a period of time, and this could include some degree of shedding on the farm where the infection originated, as well as on nearby farms. Recovery of M. avium subsp. paratuberculosis from environmental samples on sheep and goat farms in New Zealand was very low 5 months after the infected stock were removed, but infected wildlife with longer life spans may continue shedding at some level beyond this period of time (27). Environmental contamination may be further amplified by protozoa ubiquitous in soil, water and plants. Studies have noted that M. avium subspecies avium can infect Acanthamoeba castellani and be found in both protozoal life stages (trophozoite and cyst) where they are resistant to inactivation by chemical disinfectants and UV light (25). If also true for M. avium subsp. paratuberculosis, control of manure contamination of the environment should become an even higher priority in Johne's disease control programs.
Infected wildlife may have the greatest effect on the epidemiology of M. avium subsp. paratuberculosis infection on farms that have eliminated all infected livestock from the premises or on M. avium subsp. paratuberculosis-free farms in the same geographic area as infected farms. If wildlife become infected and shed the organism in a sufficient volume and in accessible locales frequented by susceptible livestock (i.e., young animals), it is possible that domestic ruminant species could become reinfected.
Studies on the pathogenesis and shedding of M. avium subsp. paratuberculosis in wildlife are needed to assess the impact of the infection on wildlife populations and the risk that infected wildlife may pose to livestock. Direct or indirect transmission among wildlife species, independent of continual contact with infected domestic livestock, might result in the establishment of carriers or local wildlife reservoirs. Subsequent transmission of M. avium subsp. paratuberculosis from wildlife back to livestock could interfere with state or national Johne's disease control programs. Comprehensive information on the duration and volume of shedding in infected wildlife could be used to calculate contamination levels under different environmental conditions and clarify the impact of this important animal infection for both captive and free-ranging species.
Acknowledgments
Primary funding for this project was provided through grant 03-9100-0802-GR, U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services. Additional funds were provided through sponsorship from 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; through the Federal Aid to Wildlife Restoration Act (50 Stat. 917) and Grant Agreement 14-45-GT09-96.0002, Biological Resources Division, U.S. Geological Survey, U.S. Department of the Interior; and through Cooperative Agreement 03-9613-0032-CA, Veterinary Services, Animal and Plant Health Inspection Service, U.S. Department of Agriculture. Ohio Agricultural Research and Development Center federal and state funds appropriated and funding through the USDA-NRICAP project on Johne's disease Integrated Program supported the genotyping studies.
We appreciate the laboratory assistance provided by Gail Thomas, Heather Cushing, Brenna Kunkel, Michael Kaffman, Megan Strother, Alifiya Ghadiali, and Kelly Anklam, plus the field assistance provided by Clay George, Brian Chandler, and Craig Okraska. Mel Pence provided assistance in locating potential study sites in Georgia. We also appreciate the hospitality of the families who allowed us to conduct the study on their farms.
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